Systems and methods for biological analysis

ABSTRACT

Provided herein are devices and methods suitable for sequencing, amplifying, analyzing, and performing sample preparation procedures for nucleic acids and other biomolecules.

CROSS-REFERENCE

This application is a national phase entry of PCT Application No.PCT/US2014/027544, filed Mar. 14, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/799,396, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/799,483, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/799,944, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/800,410, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/800,443, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/801,560, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/801,929, filed Mar. 15, 2013, eachof which applications is incorporated herein by reference in itsentirety and for all purposes.

BACKGROUND

The goal to elucidate the entire human genome has created interest intechnologies for rapid nucleic acid (e.g., DNA) sequencing, both forsmall and large scale applications. Important parameters are sequencingspeed, length of sequence that can be read during a single sequencingrun, and amount of nucleic acid template required to generate sequencinginformation. Large scale genome projects are currently too expensive torealistically be carried out for a large number of subjects (e.g.,patients). Furthermore, as knowledge of the genetic basis for humandiseases increases, there will be an ever-increasing need for accurate,high-throughput DNA sequencing that is affordable for clinicalapplications. Practical methods for determining the base pair sequencesof single molecules of nucleic acids, preferably with high speed andlong read lengths, may provide measurement capability.

Nucleic acid sequencing is a process that can be used to providesequence information for a nucleic acid sample. Such sequenceinformation may be helpful in diagnosing and/or treating a subject witha condition. For example, the nucleic acid sequence of a subject may beused to identify, diagnose and potentially develop treatments forgenetic diseases. As another example, research into pathogens may leadto treatment for contagious diseases. Unfortunately, though, existingsequencing technology of the status quo is expensive and may not providesequence information within a time period and/or at an accuracy that maybe sufficient to diagnose and/or treat a subject with a condition.

SUMMARY

Recognized herein is the need for improved devices and methods forsequencing, amplifying, analyzing, and/or performing sample preparationprocedures for nucleic acids and other biomolecules.

An aspect of the disclosure provides a method for nucleic acidsequencing, comprising: (a) directing a plurality of particles onto anarray of sensors, wherein an individual particle among the plurality ofparticles comprises a template nucleic acid molecule coupled thereto,wherein the array comprises a plurality of sensors, wherein anindividual sensor among the plurality of sensors comprises a transmitterelectrode and a receiver electrode, which transmitter electrode orreceiver electrode is shared with at least another individual sensoramong the plurality of sensors; (b) positioning the individual particleadjacent to the individual sensor; (c) performing a primer extensionreaction on the template nucleic acid molecule at the individual sensor;and (d) during or subsequent to performing the primer extensionreaction, measuring a signal that is indicative of a change in impedancebetween the transmitter electrode and receiver electrode.

In some embodiments, the primer extension reaction comprises growing anucleic acid strand that is complementary to the template nucleic acidmolecule. In some embodiments, the performing a primer extensionreaction on the template nucleic acid molecule at the individual sensormay comprise directing nucleotides or nucleotide analogs onto the arrayof sensors. In some embodiments, the nucleotides or nucleotide analogscan be directed onto the array of sensors sequentially. Moreover, themethod can further comprise (i) directing a primer onto the array, (ii)bringing the primer in contact with the nucleic acid molecule, and (iii)hybridizing the primer with the template nucleic acid molecule. In someembodiments, the at least another individual sensor can be directlyadjacent the individual sensor. In some embodiments, the at leastanother individual sensor can be separated from the individual sensor byone or more intermediate sensors of the array of sensors. In someembodiments, the at least another individual sensor may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 other individual sensors of thearray of sensors.

Moreover, the individual particle may be positioned at the individualsensor such that the transmitter electrode and receiver electrode areelectrically coupled to a Debye layer of the individual particle. Insome embodiments, the individual particle can be positioned at theindividual sensor such that at least one of the transmitter electrodeand receiver electrode is coupled with the individual particle. In someembodiments, the transmitter electrode and receiver electrode can beelectrically isolated. For example, the transmitter electrode andreceiver electrode can be electrically isolated by one or moreelectrically insulating layers. In some embodiments, the transmitterelectrode and receiver electrode are electrically isolated in theabsence of the individual particle positioned adjacent thereto.

Furthermore, the individual particle can be positioned adjacent to thetransmitter electrode and receiver electrode, thereby bringing thetransmitter electrode in electrical communication with the receiverelectrode. In some embodiments, the transmitter electrode or receiverelectrode, but not both, may be shared with the at least anotherindividual sensor. In some embodiments, the transmitter electrode can beshared with the at least another individual sensor.

Also, the method can further comprise amplifying the template nucleicacid molecule, including amplifying the template nucleic acid moleculeprior to performing a primer extension reaction on the template nucleicacid molecule at the individual sensor. In some embodiments, thetemplate nucleic acid molecule can be amplified while subjecting theindividual particle to an electric field. In some embodiments, thetemplate nucleic acid molecule can be amplified while the individualparticle is held at the individual sensor.

Additionally, the individual particle can be positioned adjacent to theindividual sensor using an electric field and/or magnetic field providedby aid individual sensor. In some embodiments, the individual particlemay be positioned adjacent to the individual sensor using an electricfield and a magnetic field. In some embodiments, the magnetic field canbe constant, and the electric field can be independently controllable toprovide (i) a net attractive force to direct the individual particle tothe individual sensor or (ii) a net repulsive force to direct theindividual particle away from the individual sensor. In someembodiments, the electric field can be constant, and the magnetic fieldcan be independently controllable to provide (i) a net attractive forceto direct the individual particle to the individual sensor or (ii) a netrepulsive force to direct the individual particle away from theindividual sensor. In some embodiments, the individual sensor isindependently addressable from other sensors in the array of sensors.

Moreover, the method can comprise measuring a signal that is indicativeof a change in impedance across (i) the individual particle or (ii) afluid environment comprising the individual particle. In someembodiments, the array of sensors may be planar. In addition, thedirecting a plurality of particles onto an array of sensors, wherein anindividual particle among the plurality of particles comprises atemplate nucleic acid molecule coupled thereto, wherein the arraycomprises a plurality of sensors, wherein an individual sensor among theplurality of sensors comprises a transmitter electrode and a receiverelectrode, which transmitter electrode or receiver electrode is sharedwith at least another individual sensor among the plurality of sensorsmay further comprise (i) flowing a fluid comprising the plurality ofparticles along a channel to the array, (ii) with the plurality ofparticles in the array, stopping or altering the flow of the fluid, and(iii) removing excess beads from the array.

In some embodiments, the method can further comprise usingJoule-heating-induced flow of a fluid comprising the individualparticle, the template nucleic acid molecule, reagents for nucleic acidamplification, reagents for the primer extension reaction, and/orproducts of the primer extension reaction, to isolate and/or concentratethe fluid at the individual sensor. In some embodiments, the particlesare nucleic acid nanoballs.

An additional aspect of the disclosure provides a system for nucleicacid sequencing, comprising: (a) an array of sensors comprising aplurality of sensors, wherein an individual sensor among the pluralityof sensors comprises a transmitter electrode and a receiver electrode,which transmitter electrode or receiver electrode is shared with atleast another individual sensor among the plurality of sensors; and (b)a computer processor that is electrically coupled to the array ofsensors and programmed to measure a signal that is indicative of achange in impedance between the transmitter electrode and receiverelectrode during or subsequent to a primer extension reaction on atemplate nucleic acid molecule at the individual sensor.

In some embodiments, the at least another individual sensor may bedirectly adjacent the individual sensor. In some embodiments, the atleast another individual sensor can be separated from the individualsensor by one or more intermediate sensors of the array of sensors. Insome embodiments, the at least another individual sensor may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 other individual sensors of thearray of sensors.

Moreover, the system may further comprise a particle that is positionedat the individual sensor such that the transmitter electrode andreceiver electrode are electrically coupled to a Debye layer of theindividual particle. In some embodiments, the particle can be positionedat the individual sensor such that at least one of the transmitterelectrode and receiver electrode is coupled with the particle. In someembodiments, the transmitter electrode and receiver electrode can beelectrically isolated. For example, the transmitter electrode andreceiver electrode can be electrically isolated by one or moreelectrically insulating layers. In some embodiments, the transmitterelectrode and receiver electrode are electrically isolated in theabsence of a particle positioned adjacent thereto.

Furthermore, the transmitter electrode or receiver electrode, but notboth, may be shared with the at least another individual sensor. In someembodiments, the individual sensor can further comprise an electricfield element and a magnetic field element. In some embodiments, themagnetic field element can provide a constant magnetic field and theelectric field element can provide an electric field that isindependently controllable to provide (i) a net attractive force todirect the individual particle to the individual sensor or (ii) a netrepulsive force to direct the individual particle away from theindividual sensor. In some embodiments, the electric field element canprovide a constant magnetic field and the magnetic field element canprovide a magnetic field that is independently controllable to provide(i) a net attractive force to direct the individual particle to theindividual sensor or (ii) a net repulsive force to direct the individualparticle away from the individual sensor. In some embodiments, theelectric field element can be integrated with the magnetic fieldelement. In some embodiments, the individual sensor is independentlyaddressable from other sensors in the array of sensors.

Additionally, the computer processor can be programmed to measure asignal that is indicative of a change in impedance across (i) theindividual particle, (ii) a Debye layer of the individual particle,and/or (iii) a fluid environment comprising the individual particle. Insome embodiments, the array of sensors is planar. In some embodiments,the system further comprises a fluid flow apparatus that is in fluidcommunication with the array. In some embodiments, the array is part ofa chip that is removable from the fluid flow apparatus. In someembodiments, the system further comprises a nucleic acid amplificationmodule and sample preparation module in fluid communication with thefluid flow apparatus, wherein the modules are removable from the fluidflow apparatus. In some embodiments, the fluid flow apparatus may be amicrofluidic device.

Another aspect of the disclosure provides an integrated point of caresystem for sensing and/or analyzing a biological sample from a subject,comprising: (a) a chip comprising a plurality of sensors as part of anarray of sensors, wherein an individual sensor among the plurality ofsensors comprises a transmitter electrode and a receiver electrode,which transmitter electrode or receiver electrode is shared with atleast another individual sensor among the plurality of sensors; (b) asample preparation module that is adapted to receive the biologicalsample from the subject and generate a processed sample; (c) a fluidflow system in fluid communication with the sample preparation moduleand the array, wherein the fluid flow system is adapted to direct atleast a portion of the processed sample from the sample preparationmodule to the array; and (d) a computer processor that is electricallycoupled to the chip and programmed to measure a signal that isindicative of a change in impedance between the transmitter electrodeand receiver electrode when the processed sample is adjacent to theindividual sensor.

In some embodiments, the at least another individual sensor can bedirectly adjacent the individual sensor. In some embodiments, the atleast another individual sensor can be separated from the individualsensor by one or more intermediate sensors of the array of sensors. Insome embodiments, the at least another individual sensor may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 other individual sensors of thearray of sensors.

Moreover, the processed sample can comprise a plurality of particleseach having an analyte coupled thereto, which analyte is generated fromthe biological sample by the sample preparation module. In someembodiments, the point of care system can further comprise a particleamong the plurality of particles that is positioned at the individualsensor such that the transmitter electrode and receiver electrode areelectrically coupled to a Debye layer of the individual particle. Insome embodiments, the particle can be positioned at the individualsensor such that at least one of the transmitter electrode and receiverelectrode is coupled with the particle.

Additionally, the transmitter electrode and receiver electrode may beelectrically isolated. For example, the transmitter electrode andreceiver electrode can be electrically isolated by one or moreelectrically insulating layers. In some embodiments, the transmitterelectrode and receiver electrode may be electrically isolated in theabsence of a particle positioned adjacent thereto. In some embodiments,the transmitter electrode or receiver electrode, but not both, may beshared with the at least another individual sensor.

Furthermore, the individual sensor may comprise an electric fieldelement and a magnetic field element. In some embodiments, the magneticfield element can provide a constant magnetic field and the electricfield element can provide an electric field that is independentlycontrollable to provide (i) a net attractive force to direct theindividual particle to the individual sensor or (ii) a net repulsiveforce to direct the individual particle away from the individual sensor.In some embodiments, the electric field element can provide a constantmagnetic field and the magnetic field element can provide a magneticfield that is independently controllable to provide (i) a net attractiveforce to direct the individual particle to the individual sensor or (ii)a net repulsive force to direct the individual particle away from theindividual sensor. In some embodiments, the electric field element maybe integrated with the magnetic field element. In some embodiments, theindividual sensor may be independently addressable from other sensors inthe array of sensors.

Moreover, the computer processor may be programmed to measure a signalthat is indicative of a change in impedance across (i) the processedsample, (ii) a Debye layer of the processed sample, and/or (iii) a fluidenvironment comprising the processed sample, when the processed sampleis disposed adjacent to the individual sensor. In some embodiments, thearray of sensors can be planar. In some embodiments, the fluid flowsystem may be part of a microfluidic device. In some embodiments, thechip can be removable from the microfluidic device. In some embodiments,the sample preparation module may comprise a nucleic acid amplificationmodule and a sample preparation module.

In addition, the biological sample may be whole blood. In someembodiments, the processed sample may comprise one or more of a nucleicacid(s), protein(s), antibody(ies), antigen(s) and cell(s). In someembodiments, the computer processor can be in a housing that is separatefrom the chip. In some embodiments, a cartridge may comprise the chipand the cartridge can be inserted into or removed from the housing. Insome embodiments, the computer processor and the chip may be in the samehousing. In some embodiments, the point of care system may be capable ofdetecting more than one of a nucleic acid, a protein, an antibody, anantigen, and a cell.

Another aspect of the present disclosure provides a computer readablemedium comprising machine executable code that, upon execution by one ormore computer processors, implements any of the methods above orelsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and a computer readable medium coupledto the one or more computer processors. The computer readable mediumcomprises machine executable code that, upon execution by the one ormore computer processors, implements any of the methods above orelsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “figure” and “FIG.” herein), of which:

FIG. 1A shows a schematic of an example sensor array. FIG. 1B shows aschematic of an example sensor array with carriers immobilized to thearray. FIG. 1C shows a schematic of an example sensor array withcarriers immobilized to the array and in contact with reagents suitablefor nucleic acid amplification. FIG. 1D shows a schematic of an examplesensor array where nucleic acid amplification occurs at each arraypixel. FIG. 1E shows a schematic example of removing reagents from anexample sensor array. FIG. 1F shows a schematic of an example sensorarray where nucleic acids are sequenced at each pixel of the array.

FIGS. 2A-2C show schematics of example carriers comprising nucleic acidnanoballs. FIGS. 2D-2G show schematic examples of nucleic acid networks.

FIG. 3 shows a schematic example of an amplification method suitable forgenerating a nucleic acid nanoball.

FIG. 4 shows an example pixel comprising a sensor of a sensor array.

FIGS. 5A-5E show schematics of various example sensor arrays.

FIG. 6 shows an example sensor array with carriers immobilized to thesensor array.

FIG. 7 is a schematic of an example method for removing an immobilizedcarrier from a sensor array.

FIG. 8 is a schematic of an example method for removing an immobilizedcarrier from a sensor array.

FIG. 9 is a schematic of an example method for removing an immobilizedcarrier from a sensor array.

FIG. 10 shows an example sensor array with carriers immobilized to thesensor array.

FIG. 11 is a schematic of an example step in an amplification method.

FIG. 12 is a schematic of an example step in an amplification method.

FIG. 13 is a schematic of an example step in an amplification method.

FIG. 14 is a schematic of an example step in an amplification method.

FIG. 15 is a schematic of an example step in an amplification method.

FIG. 16 is a schematic of an example step in an amplification method.

FIG. 17 is a schematic of an example step in an amplification method.

FIG. 18 is a schematic of an example step in an amplification method.

FIG. 19 is a schematic of an example step in an amplification method.

FIG. 20 is a schematic of an example step in an amplification method.

FIG. 21 is a schematic of an example step in an amplification method.

FIG. 22 is a schematic of an example step in an amplification method.

FIGS. 23A-F are schematics of example steps in an amplification method.

FIGS. 24A-C show schematics electrodes in an array.

FIG. 25A is a schematic of an example sensor. FIGS. 25B-25C are graphicrepresentations of operating example electrodes. FIG. 25D is a schematicof example electrodes. FIG. 25E is a schematic of example electric fieldlines generated from example electrodes.

FIG. 26 is a schematic of example electrodes in an array.

FIGS. 27A-D are schematics of example electrodes in an array.

FIG. 28 is a schematic of example electrodes in an array.

FIGS. 29A-B are schematics of example electrodes in an array.

FIGS. 30A-E are schematics of magnetic element-electrode configurations.

FIG. 31 is a schematic of example electrodes in an array.

FIG. 32 is a schematic of an example configuration of elements of anarray.

FIG. 33 is a schematic of an example configuration of elements of anarray.

FIG. 34 is a schematic of an example configuration of elements of anarray.

FIG. 35 is a schematic of an example configuration of elements of anarray.

FIG. 36 is a table that includes example magnet configurations.

FIG. 37 is a schematic of example forces that can be exerted on acarrier.

FIG. 38 is a legend for the schematic of FIG. 37.

FIG. 39 is a schematic of an example carrier coupled to a magneticelement.

FIG. 40 is a schematic of example magnetic field lines generated by amagnetic element of an array.

FIG. 41 is a schematic of example electrode configurations in an array.

FIG. 42 is a schematic of example electrode configurations in an array.

FIG. 43 is a schematic of example electrodes at a pixel of an array.

FIG. 44 is a schematic of an example array coupled to example controland readout modules.

FIG. 45 is a schematic of example electrodes of an array.

FIG. 46 is a schematic depicting an example valve system.

FIG. 47 is a schematic depicting an example valve system.

FIG. 48 is a schematic depicting an example valve system.

FIG. 49 is a photograph of an example valve system.

FIG. 50 is a schematic depicting an example valve system.

FIG. 51 is a photograph of a chip comprising a valve system.

FIG. 52 is a schematic of a step of an example method using fluidicchannels coated with hydrophobic materials.

FIGS. 53A-B are schematics of steps of an example method using fluidicchannels coated with hydrophobic materials. FIGS. 53C-D are schematicsof example manifolds.

FIGS. 54A-D are schematics of example steps of an example method to loadcarriers onto an array.

FIG. 55 is a set of photographs depicting loading of arrays withcarriers.

FIG. 56 is a set of photographs depicting loading of arrays withcarriers.

FIG. 57 is a graphic depiction of example electrode operation.

FIG. 58 is a set of graphic depictions of example electrode operation.

FIG. 59 is a graphic depiction of example electrode operation.

FIG. 60 is a set of graphic depictions of example electrode operation.

FIG. 61A is a schematic depicting an example ofdielectrophoresis-induced flow generated by example electrodes. FIG. 61Bis a schematic depicting an example of Joule heating induced flow.

FIG. 62A is a schematic depicting an example of synchronizing a DC pulsewith heat cycling. FIGS. 62B and 62C are schematics of nucleic acidscoupled to beads.

FIG. 63 is a schematic of an example system for sequencing a nucleicacid.

FIGS. 64A-B are schematics that depict example steps of an examplemethod that can be used to fragment nucleic acids.

FIGS. 65A-B are schematics that depict example steps of an examplemethod that can be used to fragment nucleic acids.

FIGS. 66A-B are schematics that depict example steps of an examplemethod that can be used to fragment nucleic acids.

FIGS. 67A-B are schematics that depict example steps of an examplemethod that can be used to fragment nucleic acids.

FIGS. 68A-C are schematics of example species that can be used innucleic acid amplification.

FIG. 69 is a schematic depicting a three-dimensional line drawing of X,Y, and Z directions.

FIGS. 70A-E are schematics of views of example microfluidic devices.

FIG. 71 is a schematic of layers of an example microfluidic device.

FIGS. 72A-C are schematics of views of an example microfluidic devicecomprising example modules.

FIGS. 73A-C are schematics of views of an example microfluidic devicecomprising example modules.

FIGS. 74A-B are schematics of views of an example microfluidic devicecomprising example modules.

FIG. 75 is a schematic of an example microfluidic device comprisingexample pins to direct flow in the device.

FIG. 76 are schematics of example pins for use in a microfluidic device.

FIG. 77 is a schematic of an example microfluidic device.

FIG. 78 is a schematic depicting an example integrated sample analysissystem.

FIG. 79 is a schematic depicting an example of aptamer-based detection.

FIG. 80A-80C are schematics depicting example sensors. FIG. 80D is aphotograph depicting an example sensor.

FIGS. 81A-B are graphic depictions of the sensitivity of examplesensors.

FIG. 82 is a schematic depicting an example sensor.

FIG. 83 is a schematic of an example control system.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “analyte,” as used herein, generally refers to any type ofbiological molecule including, for example, simple intermediarymetabolites, sugars, lipids, and hormones as well as macromolecules suchas complex carbohydrates, phospholipids, nucleic acids (e.g., DNA, RNA,mRNA, miRNA, rRNA, tRNA), polypeptides and peptides. Furthernon-limiting examples of analytes include drugs, drug candidates,prodrugs, pharmaceutical agents, drug metabolites, biomarkers such asexpressed proteins and cell markers, antibodies, serum proteins,cholesterol and other metabolites, electrolytes, metal ions,polysaccharides, genes, proteins, glycoproteins, glycolipids, lectins,growth factors, cytokines, vitamins, enzymes, enzyme substrates, enzymeinhibitors, steroids, oxygen and other gases found in physiologic fluids(e.g., CO2), cells, cellular constituents, cell adhesion molecules,plant and animal products, cell surface markers (e.g., cell surfacereceptors and other molecules identified herein as receptor proteins),and cell signaling molecules. Non-limiting examples of protein analytesinclude membrane associated proteins (e.g., extracellular membraneproteins, intracellular membrane proteins, integral membrane proteins,or transiently membrane-associated proteins), cytosolic proteins,chaperone proteins, proteins associated with one or more organelles(e.g., nuclear proteins, nuclear envelope proteins, mitochondrialproteins, golgi and other transport proteins, endosomal proteins,lysosomal proteins, etc.), secreted proteins, serum proteins, andtoxins. Non-limiting examples of analytes for detection includeAdiponectin, Alanine Aminotransferase (ALT/GPT), Alpha-fetoprotein(AFP), Albumin, Alkaline Phosphatase (ALP), Alpha Fetoprotein,Apolipoprotein A-I (Apo A-I), Apolipoprotein B (Apo B), ApolipoproteinB/Apoplipoprotien A-1 Ratio (Apo B/A1 ratio), Aspartate Aminotransferase(AST/GOT), AspirinWorks® (1′-Dehydro-Thromboxane B2), Bicarbonate (CO2),Bilirubin, Direct (DBIL), Bilirubin, Total (TBIL), Blood Urea Nitrogen(BUN), Carboxy terminal collagen crosslinks (Beta-CrossLaps), Calcium,Cancer Antigen 125 (CA 125), Cancer Antigen 15-3 (CA 15-3), CancerAntigen 19-9 (CA 19-9), Carcinoembryonic Antigen (CEA), Chloride (C1),Complete Blood Count w/differential (CBC), C-peptide, C-reactive protein(CRP-hs), Creatine Kinase (CK), Creatinine (serum), Creatinine (urine),Cytochrome P450, Cystatin-C, D-Dimer, Dehydroepiandrosterone Sulfate(DHEA-S), Estradiol, F2 Isoprostanes, Factor V Leiden, Ferritin,Fibrinogen (mass), Folate, Follicle-stimulating Hormone (FSH), FreeFatty Acids/Non-Esterified Fatty Acids (FFA/NEFA), Fructosamine,Gamma-glutamyl Transferase (GGT), Glucose, HbA1c & estimated AverageGlucose (eAG), HDL2 subclass, High-density Lipoprotein Cholesterol(HDL-C), High-density Lipoprotein Particle Number (HDL-P),High-sensitivity C-reactive Protein (hs-CRP), Homocysteine, Insulin,Iron and TIBC, Lactate dehydrogenase (LDH), Leptin, Lipoprotein (a)Cholesterol (Lp(a) chol), Lipoprotein (a) Mass (Lp(a) mass),Lipoprotein-associated Phospholipase A2 (Lp-PLA2), Low-densityLipoprotein Cholesterol, Direct (LDL-C), Low-density LipoproteinParticle Number (LDL-P), Luteinizing Hormone (LH), Magnesium,Methylenetetrahydrofolate reductase (MTHFR), Micro-albumin,Myeloperoxidase (MPO), N-terminal Pro b-type Natriuretic Peptide(NT-proBNP), Non-High-density Lipoprotein Cholesterol, Omega-3 FattyAcid Profile, Osteocalcin, Parathyroid Hormone (PTH), Phosphorus,Potassium (K+), Prostate Specific Antigen, total (PSA, total),Prothrombin, Resistin, Sex Hormone Binding Globulin (SHBG), Small DenseLow-density Lipoprotein (sdLDL), Small dense low-densityLipoprotein/Low-density Lipoprotein Cholesterol Ratio (sd LDL/LDL-Cratio), Sodium (NA+), T Uptake, Testosterone, Thyroid-stimulatinghormone (TSH), Thyroxine (T4), Total Cholesterol (TCHOL), Total Protein,Triglycerides (TRIG), Triiodothyronine (T3), T4 (free), Uric Acid,Vitamin B12, 25-hydroxy-vitamin D, clotting factors (e.g., factor I(fibrinogen), factor II (prothrombin), factor III (tissuethromboplastin), factor IV (calcium), factor V (proaccelerin), factor VI(no longer considered active in hemostasis), factor VII (proconvertin),factor VIII (antihemophilic factor), factor IX (plasma thromboplastincomponent; Christmas factor), factor X (stuart factor), factor XI(plasma thromboplastin antecedent), factor XII (hageman factor), factorXIII (fibrin stabilizing factor)).

The term “aptamer,” as used herein, generally refers to a peptide,nucleic acid, or a combination thereof that is selected for the abilityto specifically bind one or more target analytes. Peptide aptamers areaffinity agents that generally comprise one or more variable loopdomains displayed on the surface of a scaffold protein. A nucleic acidaptamer is a specific binding oligonucleotide, which is anoligonucleotide that is capable of selectively forming a complex with anintended target analyte. The complexation is target-specific in thesense that other materials, such as other analytes that may accompanythe target analyte, do not complex to the aptamer with as great anaffinity. It is recognized that complexation and affinity are a matterof degree; however, in this context, “target-specific” means that theaptamer binds to target with a much higher degree of affinity than itbinds to contaminating materials. The meaning of specificity in thiscontext is thus similar to the meaning of specificity as applied toantibodies, for example. The aptamer may be prepared by any knownmethod, including synthetic, recombinant, and purification methods.Further, the term “aptamer” also includes “secondary aptamers”containing a consensus sequence derived from comparing two or more knownaptamers to a given target.

The term “antibody,” as used herein, generally refers to immunoglobulinssuch as IgA, IgG, IgM, IgD, and IgE, whether monoclonal or polyclonal inorigin. The methods for binding and elution for the binding pairs foraffinity chromatography depend on the binding pair used, and aregenerally well known in the art. As one example, solutes withpolyhistidine labels may be purified using resins including but notlimited to commercially available resins such as Superflow Ni-NTA(Qiagen) or Talon Cellthru Cobalt (Clontech). Polyhistidine-labeledsolutes may, for example, be eluted from such resins with bufferscontaining imidzole or glycine. Buffers for ion exchange chromatographymay be selected such that the binding pair used is soluble in thebuffer. Buffers are typically single phase, aqueous solutions, and maybe polar or hydrophobic.

The term “adjacent to,” as used herein, generally means next to, inproximity to, or in sensing or electronic vicinity (or proximity) of.For example, a first object adjacent to a second object can be incontact with the second object, or may not be in contact with the secondobject but may be in proximity to the second object. In some examples, afirst object adjacent to a second object is within about 0 micrometers(“microns”), 0.001 microns, 0.01 microns, 0.1 microns, 0.2 microns, 0.3microns, 0.4 microns, 0.5 microns, 1 micron, 2 microns, 3 microns, 4microns, 5 microns, 10 microns, or 100 microns of the second object.

Integrated Sequencing Platforms

An integrated sequencing platform may include a nucleic acid (e.g., DNA)extraction system, a library construction system, an amplificationsystem, an enrichment system, and a sequencing system. In someembodiments the systems may be separate and/or in modular format. Insome embodiments, the integrated sequencing platform can include one,two, three, four, or all five of these systems. In some cases, thesystems can be integrated within a single microfluidic device and/or asingle array (e.g., a re-usable array). An example of such an integratedplatform is depicted in FIG. 63. Additional examples of such integratedsequencing platforms can be found in PCT Patent Application No.PCT/US2011/054769, PCT Patent Application No. PCT/US2012/039880, PCTPatent Application No. PCT/US2012/067645, and U.S. patent applicationSer. No. 13/481,858, each of which is incorporated herein by referencein its entirety.

In some embodiments, nucleic acid (e.g., deoxyribonucleic acid (DNA))amplification and sequencing may be performed sequentially within thesame system. In such cases, sample nucleic acid may be associated with aplurality of carriers, such as, for example, beads or other types ofparticles. In some cases, the carriers may be magnetic carriers, suchas, for example, magnetic beads or paramagnetic beads. In some cases,the magnetic carriers can be entered into an array (e.g., asubstantially planar array comprising a substantially planar substrate)of magnetic features such that the magnetic carriers are held in placeby a localized magnetic field at each position (e.g., pixel) of thearray. In some embodiments, carriers (including magnetic carriers) canbe held in place at each position of an array (e.g., a substantiallyplanar array) by electrostatic force via one or more electrodes due tothe charge of the carrier or the associated nucleic acid. In otherembodiments, the carriers can be held in place at each position of thearray by physical trenches or wells. In some embodiments, the carrierscan be held in place at each position of the array by interaction of aspecies bound to the carrier with a species bound to the array (e.g.,hybridization of oligonucleotides or via ligand-capture moiety pairs).Upon immobilization of the carriers to an array, amplification of theassociated nucleic acid and sequencing of the amplified nucleic acid canbe completed sequentially.

In some embodiments, carriers may be first entered into an array (e.g.,via flow through microfluidic channels associated with the array) andcaptured by the array. After carrier capture, sample nucleic acid may becontacted with the array (e.g., via flow through microfluidic channelsassociated with the array) and subsequently captured by the carriers.Capture may occur, for example, via nucleic acids associated with thecarriers and capable of hybridizing with the sample nucleic acid. Suchnucleic acids may also be used as primers for amplification reactionsdescribed elsewhere herein.

Alternatively, a surface of the array (e.g., sensor surface, arraysubstrate surface, etc.) may comprise means suitable for capturingsample nucleic acid, including nucleic acids capable of hybridizing withthe sample nucleic acid. Such nucleic acids may also be capable ofserving as primers for amplification reactions described elsewhereherein. Such a configuration may be suitable for amplifying andsequencing a nucleic acid in the absence of a carrier.

In some embodiments, the sample nucleic acid may be provided to an arrayat extremely dilute concentrations in order to obtain a desired ratio ofmolecules of sample nucleic acid to carrier. For example, ratios of onemolecule of nucleic acid for one carrier (e.g., bead), one molecule ofnucleic acid for two carriers, one molecule of nucleic acid for threecarriers, one molecule of nucleic acid for five beads, or less, etc maybe desired.

During amplification reactions, one or more electrodes at a sensorposition of the array may be used for concentration of reagents usefulfor nucleic acid amplification, forming a “virtual well” associated witha carrier, sensor, or substrate at the array position via an electricfield. Virtual wells can permit amplification of nucleic acids at asensor position without cross-contamination of reactants with those ofother sensors of the array. In certain embodiments, amplification withina virtual well can generate a clonal population of nucleic acidassociated with a carrier, sensor surface, or substrate associated withthe virtual well.

Nucleic acid amplification may be performed in multiple cycles ifdesired. Once a first round of amplification is completed aftercontacting an array with sample nucleic acid, an array may be washed inorder to remove any unbound amplicons and other reagents in solution.Following washing, a second round of a second round of amplification maybe completed, by contacting the array with sample nucleic acid andsubjecting captured sample nucleic acid to appropriate conditions. Whereclonal populations are generated, the sample may bind only to sites(e.g., carriers, sensor surfaces, etc.) not already comprisingamplicons, as sites with amplicons from first round of amplification maybe fully loaded amplicons. The process may be repeated for any number ofamplification cycles until capture sites are exhausted. Utilizingmultiple rounds of amplification may help eliminate double Poissondistribution problems and help ensure that each sensor site isassociated with only nucleic acid sequence, such as a clonal populationof amplicons attached to a carrier. Moreover, multiple rounds ofamplification may also help maximize the use of an array, as each roundof amplification can better ensure that all of the pixels of the arrayof occupied with amplicons for sequencing.

Moreover, during sequencing reactions, one or more of the sameelectrodes and/or different electrodes may be used to detect a reactionof interest, such as nucleotide incorporation. In some cases, sensingmay be completed using a NanoNeedle and/or NanoBridge sensor, or otherelectrical or optical sensors suitable for detection. A NanoBridgesensor may function as a pH or charge sensor, as described in U.S.Published Patent Application No. US 2012/0138460, titled “BIOSENSORDEVICES, SYSTEMS AND METHODS THEREFOR”, which is incorporated herein byreference in its entirety. A sensor (e.g., nanoneedle sensor) mayfunction as a charge, conductivity and/or impedance sensor, as describedin PCT Patent Application No. PCT/US2011/054769, PCT Patent ApplicationNo. PCT/US2012/039880, PCT Patent Application No. PCT/US2012/067645, andU.S. patent application Ser. No. 13/481,858, each of which isincorporated herein by reference in its entirety. For example, thereaction of interest may be DNA sequencing.

The detection may be based on at least one of local pH change, localimpedance change, local heat detection, local capacitance change, localcharge concentration (or change thereof), and local conductivity change,such as local conductivity change of a carrier, a nucleic acid (or otheranalyte) associated with the carrier and/or a sensor. Such measurementsmay be made by directly detecting (or detecting signals that areindicative of) a local pH change, local impedance change, local heatdetection, local capacitance change, local charge concentration (orchange thereof), and local conductivity change, such as localconductivity change of a carrier, a nucleic acid (or other analyte)associated with the carrier and/or a sensor. In some cases, detectionoccurs within the Debye length of (i) a carrier, (ii) a nucleic acidassociated with a carrier or sensor, and/or (iii) a sensor. Such asensor configuration is described, for example, in PCT PatentApplication No. PCT/US2011/054769, PCT Patent Application No.PCT/US2012/039880, PCT Patent Application No. PCT/US2012/067645, andU.S. patent application Ser. No. 13/481,858, each of which isincorporated herein by reference in its entirety.

Following the completion of sequencing, carriers/nucleic acids may bedissociated from the array, the carriers and array optionally separatedfrom bound species and washed, and either or both of the carriers andarray subsequently re-used for another round of amplification and/orsequencing. Dissociation of a carrier from the array may be completed,for example, by removal/reversal of a magnetic and/or electric fieldused to hold the carrier in place. In addition or as an alternative,fluid flow and/or other type of field (e.g., external magnetic field,external electric field) capable of exerting forces sufficient forovercoming magnetic and/or electrostatic forces used to hold a carrierin place may also be used to dissociate the carrier from an array. Wherenucleic acids are directly associated with the array, in the absence ofa carrier, the array may be treated with appropriate means (e.g.,enzymatic means, chemical means, thermal means, etc.) to remove boundnucleic acids from the array. In some cases, though, it may be desirableto remove a carrier or nucleic acid from an array prior to amplificationand/or sequencing. Such removal can be achieved in analogous fashion asdescribed above.

In some embodiments, a combined amplification and sequencing system maycomprise a magnetic array that can trap a magnetic bead or particle bymagnetic force at a plurality of the array positions. In some cases, amagnetic bead may be a paramagnetic bead. Each of the array positionsmay also comprise electrodes capable of producing electric fields and/orfunctioning as sensors. Each magnetic bead or particle can comprise aDNA segment that may be clonally amplified, for example, with the aid ofelectric fields generated by one or more of the electrodes at each arrayposition.

In some embodiments, a combined amplification and sequencing system maycomprise an array of electrodes that can trap a magnetic bead orparticle by electrostatic force at a plurality of the array positions.In some cases, a magnetic bead may be a paramagnetic bead. One or moreof the same electrodes or different electrodes at each of the arraypositions may also be capable of producing electric fields and/orfunctioning as sensors. Each magnetic bead or particle can comprise aDNA segment that may be clonally amplified, for example, with the aid ofelectric fields generated by one or more of the electrodes at each arrayposition.

An example of a combined amplification and sequencing system and use ofthe example system is depicted in FIG. 1. As shown in FIG. 1A, thesystem 100 may include an array on a substrate 101 that can comprisesensors (e.g., nanosensors) 105 sometimes in communication withmicrofluidic channels defined within the platform. Sensors 105 may beassociated with substrate 101, and substrate 101 may also be associatedwith magnetic 110 and electrode 105 and 107 elements. Magnetic beads maybe positioned over the sensors 105 by magnetic 110 or electrode 105 and107 elements. The magnetic elements may form localized magnetic fieldsand the electrode elements may form localized electric fields in orderto position a carrier at each sensor 105 of the array. Moreover, themagnetic and/or electric fields may create an area of confinement forcarriers at each position of the array.

As shown in FIG. 1B, a sample comprising DNA 140 (e.g., DNA fragments)may be conveyed into the system 100. In some cases, introduction of theDNA 140 may be via microfluidic channels associated with the array. Asshown, the array may be configured with pre-localized magnetic beads 120and the magnetic beads may be associated with primers capable ofhybridizing with DNA 140, such that DNA 140 is captured by and becomesassociated with the beads 120. The magnetic beads 120 may be positionedon the array via the magnetic elements 110 and/or electrode 105 and 107elements. Alternatively or in addition, primers may be attached, bound,or associated with a sensor at a position of the array and used to trapDNA 140 at the sensor.

As shown in FIG. 1C, reagents 160 (e.g., polymerase,deoxyribonucleotides (dNTPs), and additional primers) may besimultaneously, previously, or subsequently introduced to the array. Insome cases, introduction of the reagents 160 may be via flow throughmicrofluidic channels associated with the array, such that the reagents160 are contacted with the magnetic beads 120 via flow. Via magneticand/or electrostatic forces from the appropriate array elements, themagnetic beads 120 can be maintained in the desired position as reagents160 make contact with the magnetic beads 120 via flow.

As shown in FIG. 1D, the DNA 140 associated with magnetic beads 120 canbe clonally amplified to produce amplified DNA 145 and 155 on thesurface of the magnetic beads 120. Clonal amplification may be completedusing any suitable means including a polymerase chain reaction (PCR), aprimer extension reaction, isothermal amplification, or othertechniques.

As shown in FIG. 1E, the magnetic beads 120 in the array may be washed180, removing unbound amplicons 145 and reagents 160 in solutionfollowing amplification of DNA 140. The result is magnetic beads 120comprising clonal sets of amplified DNA 155 associated with arraypositions. Washing 180 may be completed by any suitable means, such as,for example, washing with a buffer solution at a flow rate sufficient toremove the unbound amplicons 145 and reagents 160 in solution, butinsufficient to detach the magnetic beads 120 from their respectivepositions on the array.

As shown in FIG. 1F, another aliquot of reagents 160 (e.g., polymerase,primers, etc.) and sequential cycles of individual dNTPs 185 may then becontacted (e.g., via flow) with the sensor array, permittingincorporation of the dNTPs into the amplified DNA 155 of magnetic beads120. dNTPs may be introduced in individual cycles, e.g., cycle 1=A,cycle 2=T, etc. where there may be a wash step with buffer in betweeneach cycle to help reduce the chance of contamination fromunincorporated nucleotides. Polymerase used for the sequencing reaction,may be the same type of polymerase that is used for the amplificationreaction, or may be a different type of polymerase, and can beintroduced prior to or with introduction of the dNTPs. Detection of theincorporated dNTPs during each cycle can be used to sequence theamplified DNA 155, and, thus, the original sample DNA 140. Detection mayoccur, for example, via one or both of electrodes 105 and 107. In somecases, electrodes 105 and 107 can detect nucleotide incorporation eventsby measuring local impedance changes of the magnetic beads 120 and/orthe amplified DNA (or other nucleic acid) 155 associated with themagnetic beads 120. Such measurement can be made by directly measuringlocal impedance change or measuring a signal that is indicative of localimpedance change. In some cases, detection of impedance occurs withinthe Debye length of the magnetic beads 120 and/or the amplified DNA 155associated with the magnetic beads 120.

Additional examples of combined amplification and sequencing systems,for example, may be found in PCT Patent Application No.PCT/US2011/054769, PCT Patent Application No. PCT/US2012/039880, PCTPatent Application No. PCT/US2012/067645, and U.S. patent applicationSer. No. 13/481,858, which are incorporated herein by reference in theirentireties.

In some embodiments, after amplification of sample nucleic acid ontocarriers, but before sequencing, the carriers subjected to amplificationconditions may be sorted in an enrichment system, such as, for example,an electrophoretic sorter, where sorting is achieved via electrophoreticforce applied to carriers. The electrophoretic sorter may be part of asystem used to conduct amplification and sequencing, or it may be partof a different system. In the electrophoretic sorter, null carriers(e.g., carriers without amplicons), as well as carriers subject toincomplete amplification or those comprising overly short amplicons, canbe sorted from carriers comprising the desired amplicons. Additionalexamples of enrichment systems and electrophoretic sorters are describedin PCT Patent Application No. PCT/US2011/054769, PCT Patent ApplicationNo. PCT/US2012/039880, PCT Patent Application No. PCT/US2012/067645, andU.S. patent application Ser. No. 13/481,858, which are incorporatedherein by reference in their entireties.

An electrophoretic sorter may comprise channels capable of acceptingsorted carriers. Carriers (e.g., beads) with appropriate amounts ofamplified product and with amplicons of adequate length may havesufficient charge to be pulled off to an outlet channel. Where theelectrophoretic sorter is a separate system, such carriers can becollected from the outlet channel and provided back into theamplification/sequencing system for sequencing, wherein the steps ofintroducing reagents and detecting nucleotide incorporation events mayoccur as described above.

Carriers (e.g., beads) without appropriate amounts of amplified productand/or without amplicons of adequate length may flow through theelectrophoretic sorter and, instead, be directed into a waste channel.The carriers may be collected from the waste channel and may be reusedfor another cycle of amplification or other purpose upon appropriatecleaning to remove any undesirable species. For example, carriers may bewashed with a bleaching agent, such as hydrogen peroxide, to help ensurethat no contaminants remain on the carriers so that they may be reused.

The arrays and methods described herein can be used for a variety ofapplications and detection of different biological or biochemicalmoieties in addition to nucleic acids, such as antibody-antigendetection, protein detection, cell analysis, drug-discovery orscreening, ligand, small molecules or other types of analysis. Moreover,the devices and methods described herein are not limited to DNAapplications, and may be used for reactions and analysis of interest forRNA, protein detection, small molecules, etc. or other biomolecules.

In addition to sequencing reactions and/or nucleotide incorporationevents, arrays and associated sensors may also be useful in sensingother biomolecules (e.g., oligonucleotides, proteins, small molecules,peptides, etc.) and/or reactions of interest using any of the methodsand devices described herein, including directly measuring localimpedance change or measuring a signal that is indicative of localimpedance change.

In some embodiments, a sensor may detect a nucleic acid hybridizationreaction. For example, a carrier (e.g., a bead) may be linked to anucleic acid and hybridization of the nucleic acid with another nucleicacid (e.g., a primer or oligonucleotide probe) may be detected. In someembodiments, a sensor may detect a protein-protein interaction. Forexample, a carrier (e.g., a bead) may be coupled to a protein species(e.g., antibody, antibody fragment, peptide, etc.) capable of bindingwith an additional protein (e.g., a ligand). Binding of the additionalprotein to the protein species coupled to the carrier may be detected.Binding of small molecules to species linked to carriers may also bedetected. In some cases, a plurality of detection methods may beemployed to detect a biomolecule or a biological reaction of interest.Non-limiting examples of additional detection methods include anenzyme-linked immunosorbent assay (ELISA), detection of a tag (e.g.,optical dyes, fluorescent dyes), detection of a released or generatedspecies during a biological reaction of interest, etc.

A sensor (e.g., an individual sensor) described herein may beindependently addressable. An independently addressable sensor as usedherein, can refer to an individual sensor in an array whose response canbe independently detected from the responses of other sensors in thearray. An independently addressable sensor can also refer to anindividual sensor in an array that can be controlled independently fromother sensors in the array.

Carriers

Carriers (charged or neutral carriers, magnetic or non-magneticcarriers) may be of any suitable shape, including non-spherical shapes.In some embodiments, as described above, carriers may be beads. In otherembodiments, the carrier may be a dendritic structure including adendritic structure formed by a self-assembled three-dimensional DNAnetwork. A dendritic carrier may have an enlarged surface area comparedto other carriers such as beads. Increased surface area may be useful inimproving the number of primers (and, thus, amplicons) that can beassociated with nucleic acid amplicons. Moreover, a dendritic carriermay be spherical, substantially planar, oval, or any other shape. Insome embodiments, primers may be attached to a dendritic carrier andused, for example, to capture nucleic acids from samples and, in somecases, amplify the captured nucleic acids.

In some embodiments, a nucleic acid nanoball (e.g., DNA or RNA nanoball)may be used associated with a carrier or may be used as a carrier. Anucleic acid nanoball generally refers to a nucleic acid particle withat least one dimension on the nanometer scale. The particle can be athree-dimensional particle. A nucleic acid nanoball may be created byany suitable method, such as, for example, rolling circle replicationtechniques. In some cases, a nucleic acid nanoball may be free (e.g.,not associated with a surface) or they may be bound to a surface (e.g.,surface of an array, surface of a sensor, etc.), as shown in an exampledepicted in FIG. 2A. As shown in FIG. 2A, nucleic acid nanoballs 260 arebound to surface 225. As illustrated in FIG. 2B, nanoballs 260 may bebound to a carrier such as a magnetic bead 270, which can allow forspecific placement in a desired location on an array.

In some embodiments, a nanoball may be attached to a dendritic carrieror other types of particles, such as beads. A carrier may be porous orpartially porous. If a carrier is porous or partially porous, the poresize may be of sufficient size as to permit free movement of nucleicacid (e.g., DNA), polymerase, dNTPs and other moieties useful for primerextension sequencing or other applications as appropriate. In somecases, a nanoball may be associated with another nanoball that serves asa carrier, an example of which is shown in FIG. 2C. As shown in FIG. 2C,the nanoballs 260 may be bound to a carrier nanoball 265.

In some embodiments, the nanoballs may be immobilized on surfaces suchas the surface of a sensor, surface of an electrode, surface of acarrier (e.g., bead), etc. Such a surface can have any shape such asspherical, flat, rectangular, crystalline, irregular, wells, etc. Insome embodiments, the substrate material may include, for example,silicon, silicon-based material, glass, modified or functionalizedglass, magnetic material, plastic, metal, ceramic, gels, acrylic resins,biological material, etc. Nanoballs may be attached to a surface by anysuitable method, with non-limiting examples that include nucleic acidhybridization, biotin streptavidin binding, thiol binding,photo-activated binding, covalent binding, antibody-antigen, physicalconfinement via hydrogels or other porous polymers, etc., or acombination thereof. In some cases, nanoballs may be digested with anuclease (e.g., DNA nuclease) in order to generate smaller nanoballs orfragments from the nanoballs.

In some embodiments, nanoballs may be used in nucleic acidamplification. As shown in FIG. 3, rolling circle replication may beused to amplify, to form nanoballs. A primer 310 may be bound to asingle-stranded circularized template nucleic acid 300. The circularizedtemplate nucleic acid may include identical template nucleic acidregions 305 that are separate by adaptor regions 315. A stranddisplacing polymerase 320 may be used to amplify the circularizednucleic acid template. Thus the nucleic acid template 300 may berepeatedly sequenced by allowing the primer extension reaction tocontinue for many cycles completely around the circular nucleic acidsample 300, with the strand displacing polymerase (SDP) 320 displacingthe newly synthesized nucleic acid strand 340. The rolling circlereplication may take place using a nucleic acid primer attached to acarrier, such as a bead, or a solid surface. In some embodiments, thenewly synthesized nucleic acid strand may be formed into a nanoball 360due to complementarity that may exist between the adaptor regions 315 ofthe amplified nucleic acid 340. The nanoball 360 may also be used as acarrier for DNA amplification.

Nanoballs may be fabricated of species other than DNA or RNA, such asfrom a monomer or polymer, such as polystyrene. A Nanoball, such as apolystyrene nanoball, may be dissolved subsequent to sequencing, usingan organic solvent such as acetone. Dissolution of a nanoball can freeany attached species such as nucleic acids such that they can be washedaway from the nanoball (e.g., via fluid flow). Nanoballs may be porousor made of multiple types of monomers or polymers.

In some embodiments, a nucleic acid (e.g., DNA) network may be used as acarrier or in addition to a carrier, such, as for example a bead ornanoball. A network generally refers to the folding of long singlestranded nucleic acid into a desired 2-D or 3-D structure. For example,the structure may be a rectangle, a tube, a sphere, a crystallinestructure, etc. or any other shape. Networks take advantage of thespecificity of Watson-Crick base pairing in utilizing synthetic nucleicacid “staple strands” to bind the nucleic acid in various locations inorder to form a nucleic acid network, such as, for example a DNAnanostructure. Nucleic acid networks may be generated, for example, bycombining pre-synthesized nucleic acid or oligonucleotide strands thatare designed for binding. In some embodiments, suitable amplificationmethods may be used to amplify a nucleic acid to form networks, withnon-limiting examples that include bridge amplification or rollingcircle amplification to create specific topographies.

An example of generating a DNA network is shown in FIGS. 2D-F. As shownin FIG. 2D, in some embodiments, DNA strands 200 and complementary DNAstrands 210 may be paired to form a four-prong structure 220. As shownin FIG. 2E, this four prong structure may be expanded by base pairing aplurality of DNA strands in order to form, for example, a six-prong DNAstructure 225. As illustrated in FIG. 2F, this structure 225 may bebase-paired with other DNA structures 225 in order to form a DNA network240. DNA strands may be paired such that there are binding sites forsample nucleic acid. Binding sites can be, for example, single strandedDNA sequences overhanging from the DNA network and exposable to a bulksolution. Such single stranded overhanging DNA portions may serve asbinding sites for target molecules, such as sample nucleic acid and/oramplicons that are ready for sequencing. A DNA network may be used toform a variety of larger structures of varied shapes, such as, forexample, boxes or spheres. Such larger structures may be used ascarriers. An example shape is shown in FIG. 2G, where athree-dimensional (3-D) “box” 250 is formed from a DNA network 240. Thebox 250 may be used as a carrier.

In some embodiments, peptide nucleic acid (PNA) can be used to create anucleic acid network (e.g., such as a box or sphere) which can reducethe charge associated with the network and provide for easier attachmentof sample nucleic acid molecules. Moreover, the reduction in charge mayreduce noise that can be detected by a sensor during a sequencingreaction.

In some embodiments, nucleic acid network structures can be used innucleic acid amplification. For example, the network structures may beused as carriers for binding sample nucleic acids, such that it can besubsequently amplified. Following amplification, amplicons generatedduring amplification may be also bound to the network.

Nucleic acid nanoballs and networks can include various types of nucleicacids, such as DNA, RNA, or variants thereof (e.g., circularized RNA orDNA). The nucleic acids can be single stranded or double stranded.

An advantage of using nucleic nanoballs and/or network structures ascarriers can be that they can be porous. Porosity can allow for a largesurface area that may be used to bind a greater number of molecules ofsample nucleic acid. In addition, a high level of porosity may alsoallow for good access to bulk solution, both for the purpose ofattaching sample nucleic acid and also for washing.

Array Configuration

Arrays may have varied configurations depending upon the particulardevice and/or desired performance/functionality of a device. In somecases, the pixels in an array may be regularly configured,pseudo-regularly configured, or may be configured in an irregularfashion. The shape of an array may vary. In some cases, an array may bein the shape of a rectangle, a square, a circle, a triangle, a hexagon,a staggered, wrench shaped, X-shaped, or any other shape, etc. or othershape with the pixels forming a grid comprising columns and rows.Moreover, the pixel density of an array may vary and the particulardensity may affect the array's high throughput capabilities. Pixeldensity may be increased by optimizing the configuration of electrodesin order to use available space in a more efficient manner. In someembodiments, an array may be 512×512 pixels, 1024×1024 pixels, 1024×2000pixels, 10000×10000 pixels, or of another density. Pixel pitch size mayalso vary. For example, the pixel pitch of an array may be about 1 μm,1.5 μm, 2.5 μm, 3 μm, 4 μm, 5 μm, 10 μm, 20 μm, 100 μm, etc.

Magnetic Elements, Magnetic Fields, and Magnetic Force

As described elsewhere herein, magnetic elements can be incorporatedinto a device. For example, as depicted in FIG. 4, a magnetic region 400can be associated with a particle or pixel 420 in order to retain a bead460. The magnetic region can be formed by a single magnet (where thebead can rest on it), or it can be formed by two magnets, such as, forexample, magnetic bars. These two magnets may run through the middle ofthe pixel 420, with the end of the magnets facing each other at or nearthe middle of the sensor. This configuration may create a gap (notshown) between the two magnets, near the middle of the sensor. Amagnetic force may result from this configuration, and a carrier, suchas a bead, may be retained by this force, resting on or within the gap.This gap size can be, for example, 50 nm, 100 nm, 0.25 μm, 1 μm, 1.5 μm,2 μm, 2.5 μm, etc. wide. The gap size may be optimized to allow for adesired magnetic force upon the bead. Varying parameters such as, forexample, gap size can lead to optimization of such factors as beadcapture efficiency.

Reagents such as, for example sample nucleic acid (e.g., sample nucleicacid comprising DNA template strands), DNA polymerase, primers, etc. canbe then passed over the sensor (e.g., via fluid through an associatedmicrofluidic channel) and contained by an outer electrode 440 of virtualpixel 420. This outer electrode 440 may have a negative charge orelectric potential (voltage) in order to keep the negatively chargedsample nucleic acid molecules within the area of pixel 420. The bead 460may be located between two inner electrodes 480, a portion of whichremain uncovered 490 by a dielectric layer. During amplification of thesample nucleic acid, the sample nucleic acid and reagents may beconcentrated in that region. In some cases, the voltage of the two innerelectrodes 480 can be alternated to aid in retaining species at pixel420 and/or potentially prevent bubble generation, prevent interferenceof pH modulation near the electrodes on the reaction of interest.

In some embodiments, the magnetic elements may be composed of, forexample, Ni, Fe, Co, CoPt, CrCoPt, NiCoPt, a combination thereof; oranother combination of materials. Moreover, various aspects of magnetsmay be altered in order to achieve a desired magnetic force and field.Such aspects may include, for example, the magnet material, number oflayers, thickness, length, sharpness of edges, shape, configuration etc.

A magnetic element may be composed of a paramagnetic material, forexample aluminum, platinum, etc., or any other paramagnetic material ora ferromagnetic material, for example, iron, nickel, etc., or any otherferromagnetic or paramagnetic material, or a combination of materials.In some cases, magnets may be electromagnets, permanent magnets, orelectrodes, or other different subsystems to generate electromagneticfields. In some embodiments, a magnetic region or magnetic field may begenerated via an electromagnetic structure or techniques, such as, forexample, a coil with passing current, other types of electromagneticfield generation, or via MEMS-based techniques (e.g., a MEMS-basedelectromagnetic array). FIG. 36 shows a table outlining additionalexample magnet shapes and sizes, such as for example, dot magnets andbar magnets. In some cases, dot magnets may be 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 50, 100, 500, etc. μm in width and/orlength.

The efficiency of capturing carriers or other species via a magneticarray may be tuned by positioning the magnetic array with respect toflows used to supply carriers and/or reagents to the array. In somecases, magnetic elements of an array may be oriented perpendicular toflow and such a configuration may not be optimum for maximizing captureof carriers. For example, some carriers may be captured by the magneticelements of the virtual wells during flow, and other carriers may passthrough spaces between pixels of the array. Such spaces between pixelsmay not have magnetic elements and/or the magnetic field from a nearbypixel may not be sufficiently strong to capture the carriers. As shownin FIG. 5A, a flow 500 configuration that is perpendicular to themagnetic elements 530 allows for a relatively straight path between thepixels 540 and increases the likelihood that a bead 550 will continue ona straight path through the rows of space 520 between pixels 540 insteadof over a pixel 540 where a bead 550 may be confined by a magneticelement 530.

In some embodiments, magnetic elements within the array may bepositioned at a non-perpendicular angle with respect to the input flow,such that the spaces between pixels of the array are at non-parallelangle with respect to the input flow. An array may be positioned withrespect to flow, such that its magnetic elements are at an angle. Theangle at which an array is positioned with respect to flow, for example,may be about or at least about 1°, 2°, 3°, 4, 5°, 6°, 7°, 8°, 9°, 10°,11°, 12°, 13°, 14°, 15°, 16°, 17°, 18°, 19°, 20°, 21°, 22°, 23°, 24°,25°, 26°, 27°, 28°, 29°, 30°, 35°, 40°, 45°, 50°, or more. Optimizationof the positioning of an array with respect to flow can give rise to ofhigher efficiency capture of magnetic carriers and more uniform reagentdistribution, with an example shown in FIGS. 5B and 5C. As shown in FIG.5B, when a bead 550 flows 500 through the array the chances that it willpass over a pixel 540 are greater with an angled configuration 580. Asshown in FIG. 5C, using angled configuration 580 can permit a higherloading of carriers into the array.

The positioning of magnetic elements within an array may also beoptimized to improve the association of carriers with array pixels. Forexample, a staggered arrangement of magnetic elements, as opposed to aregular grid-like pattern, may be useful in improving the association ofcarriers with array pixels. For example, as shown in FIG. 5D, eachcolumn of pixels 540 in the magnetic array may be offset with respect toalignment with the previous column. This arrangement may help toincrease bead 550 loading efficiency because of the minimization of rowsof empty space. FIG. 5E, shows how using a staggered configuration canpermit a higher loading of carriers into the array. In some cases, acombination of staggered positioning of magnetic elements and angledconfigurations of arrays with respect to flow may be used to improve thecapture of carriers into the array.

In some embodiments, a carrier (e.g., bead) may sit proximate to a dotmagnet. In some cases, a carrier may be partially or entirelyimmobilized on a surface of a dot magnet. The strength of the dot magnetmay depend on a variety of factors including: magnetic material, thenumber of layers, magnet size (e.g., thickness, width, height),direction of post magnetization (e.g., horizontal, vertical), etc. oranother factor.

In some embodiments, there may be a number of forces acting on a carrier(e.g., bead). FIGS. 37 and 38 provide examples of such forces. Twoforces which may act on a carrier, for example, may be a magnetic forceand a viscous force. The magnetic force may hold the carrier in place atits appropriate pixel on a magnetic array, and the viscous force from afluid flow can push the carrier away from its appropriate pixel on amagnetic array. In order to keep the carrier in its desired pixel, themagnetic force is generally greater than the viscous force.

In some cases, the following expression, F=∇×H_(ext)=0, may be usedestimated the force (F) on a magnetizable object, so long as the fieldsare static, and the body is non-conducting.

The magnetic flux density, B, can be calculated from the solution to theMaxwell equations for static magnets using Remanence field, B_(r), forthe block magnet. Here, it may be assumed that B_(r)=1 T and theequation may be solved for the magnetic field. Then, the magnetic forceon the iron core of 1 μm beads may be calculated.

FIG. 39 shows an example schematic of an example bead captured by a dotmagnet. As shown in FIG. 39, a three-dimensional (3-D) depiction of a 1μm bead with a 26% iron core captured by a dot magnet that has a length(L), a width (W), and a height (H). The figure also shows that the threedimensions are on an X, Y, and a Z axis. There is a gap length (G)measured from the center of the iron core to the surface of the magnet.FIG. 40 shows a schematic of magnetic field lines generated by anexample dot magnet.

In some embodiments, magnetic force exerted by a magnetic element on acarrier may depend on the distance between the carrier and the magneticelement and/or the particular geometry of the magnetic element. In somecases, the magnetic force in a horizontal direction may be substantiallyhigher than the magnetic force achieved in a vertical direction. In somecases, with respect to rectangular dot magnets, the magnetic forceexerted by the dot magnet may be higher when the direction ofmagnetization is parallel to the longer side of the rectangular dotmagnet. In some embodiments, if the thickness of the magnetic element isincreased, the magnetic force may increase slightly with respect to ahorizontal direction. In some cases, such an increase, however, may besubstantially higher with respect to a vertical direction. In someembodiments, with respect to horizontal magnetization for square dotmagnets, magnetic force exerted by the magnet may increase when the sizeof the magnetic dots is increased. With respect to verticalmagnetization for square dot magnets, the size of the magnet may beoptimized such that the magnetic force is maximized.

Electrodes and Electrode Sensors

Sensors of the present disclosure, such as nanobridge and nanoneedlesensors, can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10electrodes for sensing signals associated with species in solution, suchas signals associated with a nucleic acid sequencing reaction or thedetection of an analyte in solution (e.g., protein or antibody). Suchelectrodes can be electrically isolated and can be configured to beelectrically coupled to a species being detected. The species can be insolution, coupled to a surface of an electrode, or coupled to a particle(e.g., bead in solution). In some examples, an electrode is coupled to aDebye layer of the particle. In an example, at least two electrodes arecoupled to a Debye layer (e.g., are within the Debye layer) of theparticle. In an example, at least one electrode is touching a bead andanother electrode is touching or at least within the Debye layer of thebead. The Debye layer can have a Debye length.

The arrangement of electrodes within an array may vary, depending uponthe particular configuration desired. In some embodiments, there may beone or more transmitter and one or more receiver electrodes per pixel ofan array. A transmitter electrode generally refers to an electrode thatprovides a current and a receiver electrode generally refers to anelectrode that receives a current. In some cases, in the absence of aspecies to couple to the transmitter and receiver electrodes, electricalcurrent does not flow from the transmitter to the receiver. For example,in the absence of a particle (e.g., bead) coupled to the transmitter andreceiver electrodes, a circuit having the transmitter and receiverelectrodes is open and an electrical current will not flow from thetransmitter electrode to the receiver electrode. However, when thetransmitter and receiver electrodes are electrically coupled to theDebye layer of the particle (or other species in solution), the circuitis closed and current flows from the transmitter electrode to thereceiver electrode. In some cases, at least one of the transmitter orreceiver electrodes is “shared” between neighboring pixels. Sharing ofone of the electrodes may allow for a more efficient use of space and areduction in the number of electronic components. In some embodiments, atransmitter electrode may be shared by the beads of neighboring pixels.In some embodiments, a receiver electrode may be shared by the beads ofneighboring pixels. In some embodiments, both transmitter and receiverelectrodes can be shared by the beads of neighboring pixels. In someembodiments, the transmitter and/or receiver electrodes may beconsidered to be shared by the pixels themselves if the system does notutilize carriers, such as beads. In some embodiments, a transmitterelectrode may function as a receiver electrode depending upon, forexample, the configuration of the circuit. In some embodiments, areceiver electrode may function as a transmitter electrode dependingupon, for example, the configuration of the circuit.

In some embodiments, where an array includes electrodes that are sharedbetween neighboring pixels, the number of electrodes (E) in the arraymay be expressed as a function of the number (N) of pixels. For example,the number of electrodes (E) in the array may be equal to N, N+1, N+2,N+3, N+4, N+5, N+6, N+7, N+8, N+9, N+10, N+11, N+12, N+13, N+14, N+15,N+16, N+17, N+18, N+19, N+20, N+21, N+22, N+23, N+24, N+25, N+26, N+27,N+28, N+29, N+30, N−1, N−2, N−3, N−4, N−5, N−6, N−7, N−8, N−9, N−10,N−11, N−12, N−13, N−14, N−15, N−16, N−17, N−18, N−19, N−20, N−21, N−22,N−23, N−24, N−25, N−26, N−27, N−28, N−29, N−30, 2N−1, 2N−2, 2N−3, 2N−4,2N−5, 2N−6, 2N−7, 2N−8, 2N−9, 2N−10, 2N−11, 2N−12, 2N−13, 2N−14, 2N−15,2N−16, 2N−17, 2N−18, 2N−19, 2N−20, 2N−21, 2N−22, 2N−23, 2N−24, 2N−25,2N−26, 2N−27, 2N−28, 2N−29, or 2N−30. The expression that describes aparticular array can depend, for example, upon the particulararrangement of shared electrodes within the particular array.

In some cases, at least a subset of sensors in an array can share thesame transmitter electrode but have separate receiver electrodes. As analternative, at least the subset of sensors can share the same receiverelectrode but have separate transmitter electrodes. Such configurationcan be implemented, for example, by having the sensors in a square orrectangular grid pattern, or a hexagonal pattern.

In some situations, a given sensor of an array has at least two sensingelectrodes. One of the sensing electrodes can be a transmitter electrodeand another of the sensing electrodes can be a receiver electrode. Theelectrode scan be situated in a planar configuration. One electrode canbe situated directly below a particle and another electrode can besituated at a periphery of the particle. Both electrodes can be coupledto a Debye layer (e.g., within a Debye layer) of the particle duringsensing.

An example of shared electrodes between array pixels is shown in FIG.24A. As shown in FIG. 24A, receiver electrode 2420 is shared by twopixels of an array, wherein each pixel includes a transmitter electrode2440. As shown in FIG. 24B carriers 2400 (e.g., beads) can beimmobilized at each pixel between a shared receiver electrode 2420 and atransmitter electrode 2440 associated with a pixel, such that detectionwill happen around the carrier. FIG. 24B shows a receiver electrode 2420shared between two neighboring beads 2400 such that the receiverelectrode is used by each bead in the detection of the reaction ofinterest.

Electrode-sharing by neighboring pixels may aid in enhancing the highthroughput capabilities of an array, but may be susceptible to areduction in signal to noise ratio due to cross-talk between pixels. Thetime frame in which certain electrodes are activated may be adjusted inorder to help reduce potential cross-talk and reduce the readout rate ofthe associated array circuitry. In one embodiment, as shown in anexample of FIG. 24C, an array of electrodes may comprise receiverelectrodes 2420 and transmitter electrodes 2440, wherein one receiverelectrode 2420 is shared between two beads 2400. In some cases, theremay be ground electrodes 2460 that can be used to shield the electrodesfrom potential cross talk between neighboring electrodes. The groundelectrode may make a short path to absorb unwanted current through thebuffer. Thus, the receiver electrode may only receive the current flowfrom the bead not the current from bulk solution. In other embodiments,the system may not use beads and the current flow may come from othertypes of carriers. In another embodiment, if the system does not usecarriers, the current flow may come from the sensing element of thesystem. The transmitter electrodes 2440 in FIG. 24C are labeled T1 T2,T3, T4, and T5 in order to help illustrate that they may be activated infive time phases in order to help reduce crosstalk. For example, all ofthe T1 transmitter electrodes in the array can transmit signal duringtime phase 1 to their corresponding receiver electrodes 2420. The T2,T3, T4, and T5 transmitter electrodes do not transmit during phase 1 andmay act as ground electrodes. After the signal from the T1 transmitterelectrodes collected on the receiver electrodes 2420 and the outputsignal is generated, the roles of the T1 and T2 transmitter electrodesmay switch and time phase 2 may commence.

During time phase 2, the T2 transmitter electrodes may transmit signalto their corresponding receiver electrodes 2420 and the T1, T3, T4, andT5 transmitter electrodes can act as ground electrodes. In this manner,neighboring transmitter electrodes are not activated during the sametime period, thus allowing for a signal to the receiver electrode 2420that is less likely to be distorted by noise.

During time phase 3, the T3 transmitter electrodes may transmit signalto their corresponding receiver electrodes 2420 and the T1, T2, T4, andT5 transmitter electrodes can act as ground electrodes. In this manner,neighboring transmitter electrodes are not activated during the sametime period, thus allowing for a signal to the receiver electrode 2420that is less likely to be distorted by noise.

During time phase 4, the T4 transmitter electrodes may transmit signalto their corresponding receiver electrodes 2420 and the T1, T2, T3, andT5 transmitter electrodes can act as ground electrodes. In this manner,neighboring transmitter electrodes are not activated during the sametime period, thus allowing for a signal to the receiver electrode 2420that is less likely to be distorted by noise.

During time phase 5, the T5 transmitter electrodes may transmit signalto their corresponding receiver electrodes 2420 and the T1, T2, T3, andT5 transmitter electrodes can act as ground electrodes. In this manner,neighboring transmitter electrodes are not activated during the sametime period, thus allowing for a signal to the receiver electrode 2420that is less likely to be distorted by noise.

In some embodiments, every second, third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, etc. transmitter electrode 2440 may be activatedand any other non-activated transmitter electrodes may serve as groundelectrodes in any combination. For example electrode 1 can be turnedoff, and electrodes 2 and 3 turned on, electrode 4 turned off, etc. Inanother embodiment, electrode 1 can be turned on, electrodes 2-5 turnedoff, electrode 6 turned on, etc. where the “off” electrodes are set toground.

In addition to the configuration of electrodes within an array,adjusting electrode shape and size may allow for optimization withrespect to, for example, increasing baseline current and sensitivity. Asdescribed above, a pixel may comprise one or more transmitter and one ormore receiver electrodes. The shapes and sizes of the transmitter andreceiver electrodes may be optimized depending upon the particularfunctionality and performance of the electrodes desired.

FIG. 25D provides an illustration of example electrode embodiments andexample ranges in electrode length (0.1-5 μm), width (0.1-5 μm), height(0.1-5 μm), and separation distance (0.1-3 μm) between electrodes. Theelectrode sizes can be between 7 nm to 70 nm or between 70 nm to 700 nmor between 700 nm to 7 μm, in length, weight and depth. In the exampleshown in FIG. 25D, the electrodes are rectangular with height of 2 μm,width of 1, 2, or 3 μm, and depth of 2 μm. The electrodes are spaced 1μm apart. Moreover, in some cases, an electrode (e.g., a transmitterelectrode, receiver electrode, ground electrode, etc.) may be about 0.5μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm,5.5 μm, 6.0 μm, 7.0 μm, 8.0 μm, 9.0 μm, 10 μm, 20 μm, or more in lengthand 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm,5 μm, 5.5 μm, 6.0 μm, 7.0 μm, 8.0 μm, 9.0 μm, 10 μm, 20 μm, or more inwidth, depending on pixel pitch.

An example configuration of transmitter and receiver electrodes is shownin FIG. 26. As demonstrated in FIG. 26, a transmitter/receiver electrode2640 of a pixel may have a rectangular shape, optionally with roundedcorners. The receiver/transmitter electrode 2620 may also have arectangular shape, optionally with rounded corners, and a carrier (e.g.,bead) 2600 can rest on or proximate to one end of thereceiver/transmitter electrode 2640. As shown in FIG. 26, thereceiver/transmitter electrode 2620 may be shared between two carriers2600 of neighboring pixels. The transmitter and receiver electrodes mayhave the same, or different, dimensions.

Another example configuration of transmitter and receiver electrodes isshown in FIG. 27A. As demonstrated in FIG. 27A, the transmitter/receiverelectrode 2740 of the pixel may have a modified-wrench shape. Thereceiver/transmitter electrode 2720 may have a rectangular shape,optionally with rounded corners, wherein a carrier (e.g., a bead) 2700can rest on or proximate to one end of the receiver/transmitterelectrode 2720, and the receiver/transmitter electrode 2720 may beshared between two beads of neighboring pixels. FIG. 27B shows atop-angled view and FIG. 27C shows a top view schematic of theconfiguration in FIG. 27A. Such a configuration as shown in FIGS. 27A-Cmay improve isolation of a carrier from other carriers in an array,which may result in an increase in the current from all areas of thecarrier. This configuration may also increase the sensitivity toconductivity change.

Another example configuration of transmitter and receiver electrodes isshown in FIG. 27D. As demonstrated in FIG. 27D, the receiver/transmitterelectrode 2720 of the pixel may have a modified-wrench shape, wherein acarrier (e.g., bead) 2700 can rest on or proximate to an inner curvedportion of the receiver/transmitter electrode 2720. Thereceiver/transmitter electrode may be shared between two carriers 2700of neighboring pixels. The transmitter/receiver electrode 2740 of thepixel may have a rectangular shape, optionally with rounded corners.

Another example configuration of transmitter and receiver electrodes isshown in FIG. 28. As demonstrated in FIG. 28, the transmitter/receiverelectrode 2840 of the pixel may have an “X” shape. Thereceiver/transmitter electrode 2820 may have a rectangular shape,optionally with rounded corners, wherein a carrier (e.g., bead) 2800rests on or proximate to an inner corner portion of thereceiver/transmitter electrode 2820, and may be shared between twocarriers 2800 of neighboring pixels.

In some embodiments, a carrier may rest on or proximate a transmitterelectrode. The transmitter electrode may be shared between two carriersof neighboring pixels. The transmitter and receiver electrodes of asensor may comprise any combination of the above mentioned electrodeshapes, or any other shape, such as square, circular, rectangular,irregular, etc. In some embodiments, the shape of the electrodes may beoptimized to prevent bubble generation.

In a further embodiment, the pixel may contain a ground electrode, asshown in FIG. 29A. The ground electrode 2960 may act as a barrier,helping to shield receiver/transmitter electrodes 2920 in neighboringpixels from cross-talk from transmitter/receiver electrodes 2940. Theaddition of the ground electrode 2960 may reduce output signal that isgenerated as a result of measurement of the bulk solution instead of thearea on or near the beads 2900 where a reaction of interest, such as anucleotide incorporation reaction, may be detected. In anotherembodiment, as shown in FIG. 29B, there may be a ground line 2980, inaddition to or instead of a ground electrode.

FIG. 25A provides an example schematic demonstrating various examplesignal paths that may be detected by receiving and transmittingelectrodes 2500. C_(b) and R_(b) represent the equivalent electricalcapacitance and resistance due to a bulk (chemical buffer) solution incommunication with the electrodes, respectively. R_(DNA) is theequivalent electrical resistance due to the region in close proximity ofDNA strands 2520 fixed on a bead 2540. C_(DNA), not shown, representsthe capacitance associated with the bead and DNA strands fixed on thebead, which is effectively in parallel to R_(DNA) as a lump element.R_(DNA) is different than R_(b) due to a modified concentration ofmobile ions in close proximity and associated to the fixed DNA strands(in the Debye layer of the beads and/or DNA strands). Modulation ofR_(DNA) due to nucleotide incorporation on the template DNA strand 2520fixed on the bead 2540 can be used by a sensor (e.g., a NanoNeedle, aset of two or more electrodes, a differential amplifier, a CMOS sensor,or other types of sensors) to detect the nucleotide incorporation event,and, thus sequence of DNA strand 2520. C_(dl) is the double layer (orDebye layer, which has a Debye length) capacitance associated with thereceiving and/or transmitting electrodes 2500. In some embodiments, bycyclic injection of nucleotides, a change in impedance of a carrier,nucleic acid, and/or sensor may be measured by the sensor and can beused to identify the sequence of the DNA strand 2520. In some cases,measurement of impedance occurs within the Debye layer of the carrier,nucleic acid, and/or sensor. In other cases, all four nucleotides can beintroduced simultaneously and the signal from each incorporation eventcan be decoupled if where sensors provide sufficient detectionsensitivity and time resolution.

In some embodiments, labels may be used to amplify the amount of signalfrom a nucleotide incorporation event and a change in the impedance.Such a label can comprise a charged moiety, a physical barrier (e.g.,metallic nanoballs—platinum, gold silver, etc.), a chemical orbiochemical moiety, or a polymer-based molecule, or compound that canincrease the measured signal by the sensor. The effect on current willdepend on the particular label used and its corresponding conductivity.For example, a metal label would increase current, whereas a polymerlabel would decrease current.

FIG. 25B shows the current measured by example sensors of differentshape, such as a NanoNeedle, comprising two electrodes versus thefrequency of the applied signal during a sequencing reaction. Sincevoltage is fixed and impedance is equal to voltage over current, theplots represent the inverse of impedance measured by the sensor and,thus, changes due to nucleotide incorporation. Referring now to FIG.25B, to measure the change in resistance due to nucleotideincorporation, the sensor may operate around mid-range frequency 2510 inorder to help eliminate the effect of any capacitances betweenelectrodes. To measure the capacitive change due to nucleotideincorporation, the sensor may operate in low range frequency.

For the example shown in FIG. 25B, a frequency lower than 30 kHz wasused. In some embodiments, low frequency operating conditions may dependon the size and/or geometry of one or more electrodes of a sensor, aswell as the spacing of the transmitter and receiver electrodes. As anexample, FIG. 25C shows an example where changing the shape of theelectrodes may lead to an increase in sensitivity, as can be seen by alarger change in the percentage of current over baseline current as theincorporation of nucleotide base pairs for DNA sequencing proceeds.

At low frequencies, the double layer capacitance may dominate theimpedance and the sensitivity of the sensor to changes in resistance canbecome small. At high frequencies, the parasitic capacitance between thetwo electrodes may dominate. As current goes between electrodes, thesensitivity to changes in resistance can decrease. Therefore, based onthe electrode size and geometry, the optimum operating condition can beachieved for the highest sensitivity of the sensor.

FIG. 25E provides an example schematic of electric field lines generatedfrom electrodes 2500. The electric field lines 2560 of FIG. 25E show howthe portions of the electrodes 2500 that are the farthest from the beadmainly sense changes in the resistance or capacitance of the bulksolution 2580. The electric field lines at the portions of theelectrodes that are farthest from the bead have a direction that pointsaway from the bead, indicating that the current path is through thereagent, not around the bead. In some embodiments where the electrodesare smaller and closer to the bead, a larger portion of current goesaround the bead, which can increase the sensitivity of the sensor. Theremay be an optimum electrode configuration that increases the baselinecurrent as well as sensitivity in order to better detect nucleotideincorporation events.

In some embodiments, the frequency response of the sensors (e.g.,sensors comprising one or more electrodes) may be measured usingfrequency sweeps of the applied voltage. As shown in an example of FIG.57, the flat region of the curve (between the vertical dashed lines)represents an example frequency region that a sensor may operate in amore resistive/conductive modality when measuring sample in variedbuffer concentrations. FIG. 58 shows an example of the ratio of currentsmeasured in a sensor when samples are measured in buffers of differentconcentrations. Example optimal operating frequencies (e.g., 10 kHz˜40kHz, 30 kHz˜80 kHz, 60 kHz˜150 kHz, 300 kHz˜650 kHz, 800 kHz˜1.2 MHz,1.1 MHz˜2.5 MHz, etc.) are shown between the dashed vertical lines. Eachtrace in FIG. 58 represents an individual sensor from which measurementswere obtained. FIG. 59 displays example currents measured at variousfrequencies for sensors before bead loading 5901, after bead loading5902, and after nucleic acid incorporation events (e.g., a 61-base pairincorporation in FIG. 59) 5903. Frequency response for each of theevents 5901, 5902, and 5903 can be estimated from the plots. FIG. 60shows graphic depictions of an example of change in frequency responsedue to a reaction (right panel) (e.g., dNTP incorporation or DNAextension) or change in the fluidic environment adjacent to a sensor(e.g., bead loading) (left panel). In some embodiments, a change infrequency response can be used for detection. In some embodiments,selective frequency points can be used as a representation of a wholefrequency response for the purpose of detection (e.g., dual ormulti-frequency schemes). In some cases, there can be an optimalfrequency of operation for a given detection parameter.

In some examples, a sensor may be constructed of graphene or anothersemiconductor that has low density of states. Such materials may be usedinstead of silicon where desired and can also be used to construct aresistor. In some cases, the sensitivity of a sensor constructed of suchmaterials may be generally increased, as a small modulation of thesensor's charge may result in larger changes in its conductivity orcapacitance. In some cases, less density of states of materials used toconstruct a sensor can result in higher signal to noise ratios andsignal level. In some embodiments, a sensor may be a nanosensor, suchas, for example, a NanoBridge sensor.

Electrode-Magnet Configuration

Electrode and magnets may be arranged in a variety of configurationsdepending upon the particular device and or uses of a device desired.For example, an array may include magnetic features in order tofacilitate more efficient capture of carriers, such as for examplebeads. In some embodiments, there may be one bead associated with eachpixel, and each pixel may have one electrode and one magnetic element.The electrode and magnet may be configured such that the electrode islocated on top of the magnetic element or below the magnetic element. Insome embodiments, an array pixel may comprise an electrode with amagnetic element located underneath the electrode. A carrier (e.g.,bead) may rest on top of the electrode-magnet structure.

In some cases, the magnetic element may be covered by a thin layer ofmaterial, such as, for example, a thin layer of dielectric material,gold, and/or platinum. Such a layer of material may help to reducecorrosion of the magnet that can occur due to exposure to thesurrounding environment, such as for example buffer conditions in thesolution.

In some cases, an adhesion layer may be deposited below or on a magneticelement prior to its deposition on an array. The adhesion layer mayhave, for example, a “bar” shape. The adhesion layer may consist of, forexample, Chromium, Titanium, or another adhesive material. This adhesivelayer may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20nm, or more in thickness or may be another thickness. The bar may bemagnetized through sputtering of a magnetic layer. The magnetic layermay consist of, for example, iron, nickel or cobalt, or combinationsthereof; or any other magnetic material. The sputtered magnetic layermay be more or less than, for example, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm,30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm,400 nm, 410 nm, etc. in thickness.

FIG. 30 shows various examples of magnetic element-electrodeconfigurations. As shown in FIG. 30, exemplary embodiments include, butare not limited to: (A) A magnet 3070 covered by a thin dielectric layer3020 with an electrode layer 3040 on top of dielectric layer 3020. Insome embodiments, electrode 3040 may have a greater length than thedielectric layer 3020 and/or the electrode 3040; (B) Some portion ofmagnet 3070 may be covered directly by electrode 3040 with no dielectriclayer 3020; (C) Magnet 3070 may be covered by dielectric layer 3020, andelectrode 3040 can be smaller than the magnet 3070 that rests on top ofdielectric layer 3020 (D) Electrode 3040 may cover all of magnet 3070,with no dielectric layer 3020 associated with the structure; (E) Magnet3070 may have electrode 3040 directly on top where electrode 3040 maycover all of magnet 3070 and the thin dielectric layer 3020 can besandwiched between electrode 3040 and magnet 3070.

In a further embodiment, there may be other electrodes associated withthe pixel in addition to an electrode-magnet layered structure, anexample of which is shown in FIG. 31. FIG. 31 shows an example array ofpixels, wherein each pixel comprises a modified-X shaped electrode 3140Aand a magnetic element 3070 (represented by the dashed lines) locatedunderneath the electrode 3140A. Electrode 3140A is proximate to bead3100. Rectangular electrodes 3140B and ground electrodes 3160 are alsoassociated with each pixel. The modified X-shape electrodes 3140A may betransmitter electrodes, or they may be receiver electrodes. In a furtherembodiment, the rectangular electrodes 3140B may be the transmitterelectrodes, or they may be the receiver electrodes.

In some examples, as shown in an example side view in FIG. 32, themagnetic element may be a bar magnet (represented by a black box in FIG.32), in some cases with a long rectangular shape or other shape. Themagnetic layer may be used as a connection via between CMOS electronicsand a post-CMOS electrode. In some cases, the bar magnets of the arraymay have a small gap in between magnets where carriers (e.g., beads) maybe captured by the magnetic element.

In some examples, as shown in a side view in FIG. 33, a magnetic elementin an array pixel may be a dot magnet. In such cases, a carrier (e.g.,bead) may be captured by the magnetic element such that it is locatedproximate to a single dot magnet. Moreover, a dot magnet of a pixel maybe proximate to one end of a receiver electrode. In some cases, a dotmagnet may be located between a transmitter and a receiver electrode.The transmitter and receiver electrodes can be connected to CMOSelectronics through vias or another suitable route.

FIGS. 34 and 35 show additional configurations of dot magnetic elementsan electrodes. FIG. 34 shows a top view of an example dot magnetconfiguration where the dot magnet is located proximate to one end ofthe receiver/transmitter electrodes. FIG. 35 shows a top view of a dotmagnet configuration where the dot magnet is located between thetransmitter/receiver and receiver/transmitter electrodes.

Row/Column Multiplexing

The output signal from an individual receiver electrode may be measuredindividually, but potential problems can arise, however, with this typeof configuration because the high data readout rate that results fromthis setup may place excessive demands on a readout system. Furthermore,such a setup may lead to an increased number of electronics, such as forexample, electrical lines connecting each receiver electrode to theoutput circuitry. In some embodiments, the receiver electrodes may bemultiplexed to allow for a reduced readout rate, increased signal tonoise ratio as a result of reduced cross-talk, and fewer electronics.

FIG. 41 shows, in one embodiment, an array where there is multiplexingwith respect to both rows and columns in the array. In some embodiments,the receiver electrodes may be multiplexed according to theircorresponding column or row. Thus, the output signal can be read on aper column or per row basis, as opposed to the readout associated withindividual sensors. In a further embodiment, the receiver electrodes maybe configured such that every 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th),7^(th), 8^(th), 9^(th), 10^(th), 11^(th), 12^(th), 13^(th), 14^(th),15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th), n^(th), etc.receiver electrode or all the receiver electrodes in a column or row areconnected.

In some embodiments, all the receiver electrodes in a column or row mayshare the same electronics. In some cases, by transmitting only on onerow or column, then only the receiver electrodes on the active rows orcolumns will detect signals. Close proximity of receiver electrodes inthe same column or row, however, can result in increased cross-talkcurrent between receiver electrodes. In some cases, a transmitterelectrode in one row may transmit. In such cases, the receiverelectrodes from adjacent rows as well as the active row can contributeto the receiver signal. To mitigate and reduce receiver electrodecrosstalk, every other receiver electrode in the same column may beconnected to the same receiver line. In some embodiments, every 2^(nd),3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th), 10^(th),11^(th), 12^(th), 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),19^(th), 20^(th), n^(th), etc. receiver electrode may share the samereceiver line. In some cases, receiver lines from the same column or rowcan be multiplexed into a single receive circuitry without impactingreadout rate. Furthermore, in cases where the same receiver circuitry isused across multiple columns or rows, the result can be a reduced numberof receive electronics, sometimes at the expense of reduced readoutrate.

An example configuration of electrode multiplexing is shown in FIG. 41.As shown in FIG. 41, beads 4100 share the same receiver electrode 4140and the receiver electrodes 4140 are configured such that everyelectrode in a column is connected to the same line. This is illustratedin the figure by an “X” designation each time a receiver electrode 4140is connected to the line. Optionally, ground electrodes 4160 may be usedin order to help reduce crosstalk between pixels.

In a further embodiment, in FIG. 41 the transmitter electrodes 4120 maybe multiplexed on a per row basis. The transmitter electrodes may beconfigured such that every 2^(nd) (shown) transmitter in a row isconnected to the same line, or in other embodiments every 3^(rd),4^(th), 5^(th), 10^(th), 20^(th), n^(th) etc. transmitter electrodes maybe connected to further reduce the crosstalk between pixels.

An example configuration of electrode multiplexing is shown in FIG. 42.As shown in FIG. 42, beads 4200 sharing the same receiver electrode 4240and the receiver electrodes 4240 are configured such that every 4^(th)electrode in a column is connected to the same line. This is illustratedin the figure by an “X” designation each time a receiver electrode 4240is connected to the line. For example, in FIG. 42 when each electrode“2” is connected to the same line, this is designated by “XX”, everythird electrode connection is designated by “XXX”, etc. In a furtherembodiment, when each receiver electrode “2” is activated, receiverelectrodes “1”, “3”, and “4” may be used as ground electrodes.Optionally, ground electrodes 4260 may be used in order to help reducecrosstalk between pixels.

In a further embodiment, in FIG. 42 the transmitter electrodes 4220 maybe multiplexed on a per row basis. The transmitter electrodes may beconfigured such that every 2^(nd), 3^(rd), 4^(th), 5^(th), 10^(th),20^(th), n^(th) etc. transmitter electrodes are connected.

In some embodiments, as shown in FIG. 42, the transmitter electrodes4220 may be connected such that every fifth electrode is connected tothe same line. Multiplexing and a reduction in cross-talk may beachieved by, for example, activating electrodes (e.g., T1 electrodes inFIG. 42) on the line first during time phase 1 and measuring the signalfrom nucleotide incorporation events via the receiver electrode 4240. Atthis time, electrodes on another line (e.g., T2 electrodes shown in FIG.42) may be grounded. After the signal detection is complete, the T1electrodes may be grounded and time phase 2 may commence wherein the T2electrodes may be activated such that the incorporation event on oraround the other bead can be detected. The pattern may be continued(e.g., T3 electrodes activated during time phase 3, T4 electrodesactivated during time phase 4, T5 electrodes activated during time phase5) for the T3, T4, and T5 electrodes. An example of current flow in anindividual pixel is shown in FIG. 43. As shown in FIG. 43, the path ofthe current 4305 travels from the activated transmitter electrode 4320to the activated receiver electrode 4340 as well as to the groundelectrodes 4360 and receiver electrodes set to ground 4340G.

In some embodiments, individual rows and/or columns may be activated viacircuitry on the periphery of the array. An example of an array incommunication with periphery electronics is shown in FIG. 44. As shownin FIG. 44, a signal generator 4430 sends a signal to a signal modulator4435 and finally to a multiplexer 4445. Column multiplexing may becontrolled via a column selector 4450 and the row multiplexing may becontrolled via a row selector 4455. The row and/or column selectors mayselect one or more of the rows and/or columns, respectively, to beactivated while the other rows and/or columns are inactivated or set toground. The data output from the rows and columns may be sent todemultiplexer/readout circuitry 4465.

In some embodiments, ground electrodes as well as inactivatedtransmitter and receiver electrodes may be used to reduce cross talk andnoise. This reduction in cross talk and noise may be achieved bypreventing current from travelling from activated transmitters toreceiving electrodes outside of their own pixel. Using thisconfiguration, the activated receiver only receives current from itscorresponding transmitter. In addition, undesired current through a bulksolution in contact with a pixel may be minimized by using a groundconfiguration. In some embodiments, using this configuration, most ofthe current 4505 through the bulk solution has a shorter path to theground electrode rather than to the receiver electrode of a neighboringpixel 4540N. Thus, in some cases, most of the current 4505 detected bythe receiving electrodes 4540, therefore, can come from a bead 4500 andthe incorporation events around the bead 4500, as shown in an examplearray of FIG. 45. The ratio between the current due to incorporationevents to the base sensor current can increase, which can simplify theelectronics that may be sufficient for detection of incorporationevents.

In another embodiment, multiplexing may be synchronized by the flow ofbuffer and reagents. This may decrease the reading time required by thesystem.

Reusability & Monitoring

In some embodiments, amplification and/or sequencing arrays may bereused by the removal of carriers (e.g., beads) from the array. Removalmay be done, for example, by the application of an external magnetfield, which may result from the movement of a permanent magnet or theactivation of a magnet, to pull, move or dislodge carriers from whereinthey are held in an array. Alternatively or in addition, carriers may bereleased through chemical and/or enzymatic means.

After removing the carriers from an array, it may be possible to reusethe array by bringing new carriers (or regenerated carriers previouslyremoved) into the array, often again in a one to one correspondence withsensors in the array. In some embodiments, reusability of an array canbe monitored or controlled by counting the number of times the array isused. Counting may be accomplished in various ways. For example, if anarray uses electromagnets, the number of times the array is used can bedetermined from electrical control of the magnet. In another example, asensor (e.g., an electronic sensor) can be used to count the number oftimes the array has been used. In another example, monitoring aparticular species (e.g., a primer or adaptor) used for systemoperation, amplification, and/or sequencing can be used to count thenumber of times the array has been used. In another example, a specificbuffer, reagents, etc. with or without a specific “starting primer” or“starting adaptor” can be used.

A reusable amplification and sequencing system may provide a number ofadvantages. The cost of sequencing has a number of parts. For sequencingusing electronic sensors, one of the major costs is the cost of theprocessed silicon itself, that is, the sensor. This may be particularlytrue if the sensor is not re-useable. The magnetic and/or electrostaticarray design may allow for reuse without the need for wells fairly, asnucleic acids or other species can be configured not to be bound (e.g.,attached a carrier such as a magnetic bead) to a sensor, and thecarriers can be easily removed, for example, by reducing or removing themagnetic field which holds the carriers in place. In other embodiments,beads may be removed by increasing the flow rate or using other types offields (e.g., electric).

Selective Bead Retention/Removal

In certain situations, it may be desirable, to be able to selectivelyretain or repel one or more carriers, such as beads, as shown in FIG. 6.Such methods, as described below, may allow for selective targeting ofcarriers to pixels. In some embodiments, carriers that are bound to acertain biomolecule (e.g., nucleic acid) of interest may be selected andeither retained or removed at appropriate position(s) of an array asdescribed below.

In some embodiments, carriers may be selectively removed from an arraypixel by modulation of the magnetic field at the desired pixels. Forexample, as shown in FIG. 7, a magnetic element associated with a pixelmay comprise an electromagnet and the electromagnetic field may bereduced or turned off to a release an associated carrier (e.g., a beadas shown in FIG. 7). In some cases, the electromagnet may comprise amagnetic core surrounded by a looped, electrically conductive material.The electrically conductive material may be insulated from the core. Insome cases, the magnetic element may be ferromagnetic or paramagnetic.Where a paramagnetic magnetic element is used, a magnetic field may beinduced in the magnetic element through the application of an externalmagnetic field.

In another example shown in FIG. 8, an electrostatic dislodging orrepelling force may be used to dislodge a carrier from a desired arraypixel. A pixel may be configured such that one or more electrodes arelocated underneath the carrier (e.g., a bead shown in FIG. 8). Chargeexerted by the electrode may be reversed, increased, decreased, orturned on and off selectively depending on the desired effect on thecarrier. The electrode may have, for example, a strong negative charge,and such strong negative charge repels the associated carrier away, asshown in FIG. 8. Electrodes used to dislodge a bead from a pixel may bethose suitable for use in amplification and sequencing devices/methodsdescribed elsewhere herein.

In some cases, modulation of magnetic elements and/or electrodes at apixel as described above may be combined with fluid flow through thearray to dislodge certain beads. For example, the flow can be used afterturning off or reducing the magnetic force on the associated carrier orafter applying a dislodging force via an electrode. An example of aresult of selective removal of carriers from the array shown in FIG. 6is shown in FIG. 10. As shown in FIG. 10, desired beads are retained indesired pixels, with the rest of the beads removed from the array.

Methods for selective removal and/or retention of carriers may be usedfor identifying a molecule with a desired sequence from a pool ofmolecules in a sample. Such identification may be useful in identifyingspecies that can be used for long DNA synthesis. Synthesizing longpieces of DNA can be important in a number of biotech applications.Constructing longer DNA typically includes the construction of longerpieces via the assembly of shorter pieces or by using shorter pieces tointroduce desired changes into preexisting longer pieces of DNA. Thus,identification of such shorter molecules with desired sequence from apool of molecules in a sample can be useful in synthesizing long DNA.High-throughput sequencing provides a means for rapidly screeningthrough pools of synthesized DNA molecules. If DNA can be directlyrecovered from a specific location of an array after sequencing, it maybe possible to avoid many challenges (e.g., pipetting robots,bar-coding, cloning, etc.) associated with current approaches that usehigh-throughput or low-throughput sequencing methods now used tovalidate pools of synthesized molecules. The ability to choose aspecific bead (with a specific DNA sequence) can allow forsequence-based targeted analysis.

In some embodiments, selective removal and/or retention of carriers maybe used for more efficient and/or more selective carrier washing.Furthermore, selective removal and/or retention of carriers may enhancethe effects of dislodging carriers via fluid flow, since fluid flow maynot always be sufficient to remove carriers from the array.

Valve Systems and Minimizing Dead Volume

In some embodiments, it may be desirable to integrate a valve system aspart of a flow cell, such as for example a sensor array in communicationwith flow channels, such as, for example, microfluidic channels. A valvesystem can enable the flow of samples and other materials to varioussections of a flow cell, such that different samples may be used indifferent sections of a flow cell. In some cases, a valve system may beintegrated adjacent to a flow cell, whereby the valve system and flowcell may form a sealing interface with each other. In some embodiments,a valve system and a flow cell can be located proximate to each other onthe same mount, such as for example, the same microfluidic chip. A valvesystem can also include one or more waste valves such that fluids may beremoved from the valve system prior to flowing into various sections ofa flow cell. For example, if there is a significant amount of deadvolume in the valve system, it can be desirable to remove fluid whichmay have an unacceptable level of cross contamination (e.g., reagentsfrom a previous cycle) from a previous fluid.

In some embodiments, it may be desirable to integrate a valve devicewith the flow cell such that there may be various input channels, whichcan include inputs for reagents. For example, the various input channelsmay include channels for the four dNTPs (e.g., for DNA sequencingreactions), one or more buffer channels, salts channels, enzymechannels, and channels for other moieties that may be used for a desiredreaction, such as the incorporation of nucleotides. Input channels mayalso be employed for various buffers and wash reagents, polymerasecontaining buffers, which may also contain salts and any other moietiesneeded for polymerization, reagents needed to strip any coatings fromthe flow cell, reagents which may be needed to re-coat the flow cell,buffers which also include a phosphatase, or other reagents.

In some embodiments, the valve device may be fabricated frompolydimethylsiloxane (PDMS). In another embodiment the valve device canbe fabricated from glass with magnetically or pneumatically activatedelastomeric valves. In some embodiments, it may be desirable to bond avalve and a fluidics PDMS manifold to a silicon device. It can bedesirable to increase the bonding strength between the PDMS and thesilicon device, for example, to promote stability in the overallstructure. In some embodiments, it may be desirable to use plasmaactivated PDMS to improve bond strength. As plasma treatments which havetoo much power or too much pressure may actually decrease the bondstrength of PDMS to silicon, lower power levels and pressures can beused to address this potential issue. In one embodiment, it may besuitable to use a pressure between 500 mili Torr and 30 miliTorr and apower level between 10 and 60 watts while using, for example, a 790series Plasma-Therm.

For a device fabricated of PDMS or other similar materials, it can bepossible to use several pressure valves to control the flow of reagents.With such valves it is possible to have several valves in closeproximity to each other, and the valves may be very close to a centralchannel, reducing dead volume, an example of which is shown in FIG. 46.As shown in FIG. 46, which shows a reagent valve system 4600 with threereagent input lines 4602 with valves 4606, each of which can beconfigured to flow towards the input to a flow cell 4608, under thecontrol of pressure control lines 4604.

For a more complex system, where more reagent inputs are desired, thesimple valve system 4600 of FIG. 46 may be insufficient, as it has butthree reagent inputs lines 4602. In alternative embodiments, as shown inexamples of FIGS. 47 and 48, many more inputs can be enabled in adevice. Such an approach can also permit clearing of dead volume withina channel. For the example valve system shown in FIG. 47, inputs caninclude input ports for dATP, dTTP, dCTP, dGTP, a first buffer, a secondbuffer, and sample. Output port can include a first waste output port, asecond waste output port, and a third waste output port. Control linescan be in place for each input and output port, with additional controllines to control the direction of flow between activated ports. A wasteport is shown immediately prior to the flow cell, so that any remnantreagent from a previous flow may be removed, allowing a clean transitionfrom one reagent to another, without diffusion from any dead volumes inthe valve system.

FIG. 48 depicts an example valve system with an oval flow path, suchthat all input valve port positions have a path to an outlet (waste)port in both directions from an input valve port position. Valves asshown in FIG. 46 may be used for each valve systems shown in FIGS. 47and 48 or in an embodiment of a reagent valve system as shown in FIG.49, wherein a photograph of an example PDMS valve system is shown.

Dead volume may generally refer to one regions in the channels (e.g.,microfluidic channels) and/or chambers of a flow cell that may need tobe washed between cycles in order to remove contaminants. In someembodiments, the dead volume may be a region located between a valvesand a channel leading to a flow cell, as shown by the schematicdepiction of an example valve system in FIG. 50. As shown in FIG. 50,the valve system 5000 may include buffer input channels (B1 and B2) aswell as reagent input channels (A, C, T, and G). Also shown is a sensorchannel 5040 that can lead to a flow cell (not shown). A valve functioncan be performed by control lines that open or close their correspondingchannel. For example, control line B1C can regulate the flow to/from theB1 input channel, B2C can regulate the flow to/from the B2 inputchannel, and AC, GC, TC, CC, can each regulate the A, G, T, and C inputchannels, respectively. A central channel 5020 can connect all of theinput channels. The volume that encompasses the central channel 5020 upuntil the point where the sensor channel 5040 begins may be consideredthe dead volume (shown by dashed lines) of valve system 5000. In somecases, this volume may be washed between reagent cycles in order toprevent contamination.

In some cases, depending on the location of the valves, the dead volumemay be calculated from the reagent input location to the flow cell ifthe valves are located substantially in the same location as the reagentinput.

In some embodiments, in order to reduce dead volume, an input systemwith multiple inlet valves may be placed directly on a chip comprising aflow cell, as shown in an example system in FIG. 51. As shown in FIG.51, a valve system is located directly on a chip 5100. On-chip placementmay allow for a reduction in dead as it can allow for minimizing thedistance between the valves or the distance from the input location tothe flow cell. A reduction in dead volume may allow for a more efficientsystem and may also prevent waste of reagents.

Constant Flow and Stop-Flow Carrier Methods for Carrier Loading

In some cases, it may be desirable to provide small volumes to an arrayvia flow, such as, for example, picoliter volumes. For injectingpicoliter amounts of amplification or sequencing reagents into a fluidicsystem, a magnetic array may utilize microfluidics. For example, themicrofluidic platform may contain lines for injecting/deliveringreactants to pixels of the array. For sequencing embodiments, themicrofluidic system may be used to control sequential injections ofdNTPs to appropriate species within the array, such as the arraysubstrates or carriers immobilized to an array via localized magneticfields.

In some cases, carriers (e.g., beads) may be supplied to an array viainjection of beads into microfluidic channels of a magnetic array at aconstant flow rate. In such cases, the flow rate generally needs to befast enough for the process to be efficient, yet slow enough to allowfor the beads to be immobilized at pixels. In some embodiments, it maybe desirable to have one carrier immobilized per pixel. In someembodiments, beads may be flowed into a chamber at a substantiallyconstant rate to load the beads onto an array. FIG. 55 shows an examplearray of sensors before bead loading (left) and the array of sensorsafter bead loading with single beads. FIG. 56 shows, an example of beadloading onto an array of a microfluidic chip. Each bright white spotrepresents the presence of a bead. This example shows that the chip maybe loaded with more than 90% single beads.

In some cases, constant flow may be insufficient at supplying carriersto an array. One problem that may arise with this technique is that dueto constant flow, many carriers may never drift down far enough toeither come to rest in a pixel or, in the case of magnetic nanosensorarrays, come close enough to be in the range of the magnetic field of apixel.

Stop-flow techniques may be used to overcome the challenges of constantflow techniques and can improve loading efficacy and efficiency. FIG.54A-D illustrate an example stop flow technique. FIG. 54A shows aschematic of an example side view of a microfluidic channel 5420 whereinan array of pixels 5430 is located at the bottom of channel 5420. Asshown in FIG. 54A, a stop-flow methods may comprise flowing a solutionthat contains the carriers (e.g., beads 5410) into the channel 5420proximate to the pixels 5430 of the array. The flow 5400 of solution maybe set to flow at a constant rate.

Next, as illustrated in FIG. 54B, once the carriers 5410 have enteredchannel 5420 and traveled directly above or proximate to the array ofpixels 5430, the flow is stopped. The sudden stop in flow may allow moreof the carriers to drift down to the pixels than may otherwise bepossible using a conventional loading technique. In an example, the flowcan be stopped suddenly by terminating power to a fluid flow device(e.g., pump) and/or suddenly inducing the flow of a fluid along adirection that is opposite that of the fluid having the carriers. Insome cases, an electric and/or magnetic field can be used to immobilizethe carriers when the flow of fluid is stopped. As shown in FIG. 54C,shows a number of carriers 5410 have drifted down to settle in a pixel5430. Following settling of the carriers 5410, a wash solution may beflowed 5400 into the microfluidic channel 5420 in order to wash off anyexcess carriers 5450 that have not settled within a pixel 5430. The flow5400 generally has sufficient velocity in order to wash out the excesscarriers 5450, but not such a high velocity that it removes pixel-boundcarriers 5440 from their position in a pixel 5430. An electric and/ormagnetic field can be used to retain the carriers within the pixel 5430.FIG. 54D shows that the result of this process may be a higher carrierfill efficiency with, in some cases, one pixel-bound carrier 5440 perpixel 5430.

In some embodiments, an initial carrier (e.g., bead) loading step may beperformed at a constant flow rate. In other embodiments, an initialcarrier loading step may be performed at varied flow rates. In someembodiments, as an alternative to or in addition to stopping flow, thedirection and/or flow rate of a fluid comprising carriers may be alteredor alternated to allow for improved delivery of carriers to an array. Insome embodiments, excess carriers may be washed off of an array bywashing with solution, such as, for example a buffer solution. As analternative or in addition, excess beads may also be removed bymagnetic, electrical, physical, chemical, etc. or any other suitableremoval methods.

In some embodiments, a nano or micro-scale nebulizer may be used to helpspread beads down to the bottom of a microfluidic chamber and towardsthe pixels of an array in the chamber. The nebulizer may be located ator proximate to the top of the microfluidic chamber.

In some embodiments, one method for washing the carriers (e.g., beads)may be through the use of larger carriers (e.g., beads) flowed throughthe microfluidic channel in order to loosen/and or knock the carriersoff of the array. Some examples of materials that the larger beads maybe composed of include glass, metal, plastics or polymers, acrylic,nylon, etc. or any other material. In other embodiments, the carrierbeads may be washed using other beads that are the same size or smaller,but have sufficient velocity or mass such that the carrier beads may beloosened or removed from the array.

Hydrophobic Materials in Channels

Various microfluidic systems use valves to control the flow of solutionthrough microfluidic channels. In some instances, there may be a need toreduce the number of valves in the system and/or provide an additionalmeans of stopping of retarding fluidic flow, including at specifiedtime. A hydrophobic layer may be deposited on some portion of the innersurface of a microfluidic channel in order to allow for the regulationof flow through the channel. In some embodiments, a microfluidic channelor some portion of it may be partially or entirely composed ofhydrophobic material. In some embodiments, a hydrophobic material may bedeposited around the circumference of a microfluidic channel, creating ahydrophobic “band.” The diameter of the hydrophobic band may depend onthe diameter of the microfluidic channel. In some embodiments, ahydrophobic material may be deposited on more than one wall or portionof a microfluidic channel. The hydrophobic material may be depositedusing layer by layer (LBL) deposition or any other suitable method.

Depending on its velocity, when the fluid reaches a hydrophobicmicrofluidic channel or the hydrophobic portion of a microfluidicchannel, the flow may be stopped due to the interaction at the boundarylayer between the fluid and the hydrophobic material. An example ofhydrophobic coatings used to alter flow is shown in FIGS. 52 and 53A-B.As illustrated in FIGS. 52, 53A, and 53B, the hydrophobic material 5340may be deposited on a portion of the bottom of the microfluidic channel5300. As illustrated in FIG. 52, a portion of the bulk solution 5320Amay be separated from another portion of the bulk solution 5320B by thehydrophobic material 5340. The area of separation 5380 is shown usingdashed lines. A relatively small rate of flow 5310A may be present, butmay not be sufficient to overcome the pressure required to passhydrophobic portion 5340. In some embodiments, the bulk solution 5320Amay contain reagents that differ from those in bulk solution 5320B (orbulk solution 5320B) and in this manner the hydrophobic portion 5340 mayact as a passive valve, separating the solution.

Following, reagent retardation via hydrophobic material 5340, higherflows may be used to push the reagents through the channel. As shown inFIG. 53A, a higher rate of flow 5310B may be used to overcome thehydrophobic portion 5340 such that there is no longer a fluidicseparation area 5380. In this manner, bulk solution 5320A may passhydrophobic area 5340.

The fluidic separation area 5380 results due to the interaction betweenthe hydrophobic area 5340 and bulk fluid 5320A/B, an example of which isshown in FIG. 53B. As show in FIG. 53B, a side view of microfluidicchannel 5300, the interaction between the fluid 5320A/B and thehydrophobic material 5340 increases the contact angle 5360 of the fluid5320A as it reaches the hydrophobic interface, resulting in a separationof fluid 5380 in the channel. In this manner, the hydrophobic materialmay be used as a passive valve for regulating flow. For a channel of thesame diameter, the pressure required to pass the hydrophobic area isgreater than the pressure required to pass a non-hydrophobic portion. Assuch, the flow may be resumed upon application of a greater pressure tothe fluidic input and this may be sufficient to overcome the hydrophobicportion of the channel, resulting in flow across the hydrophobicmaterial and no separation of the fluid.

The location of passive valves in the microfluidic system may depend onthe overall configuration desired. In some embodiments, there may be amanifold leading to the input of a flow cell, so that any reagentsremaining from a previous use of the manifold may be removed, an exampleof which in FIGS. 53C and 53D. FIG. 53C shows the fluidic separationareas (passive valves) 5380 where the hydrophobic material is locatedand that a small fluid flow 5310A may be insufficient to “open” thepassive valve. A sufficiently large fluid flow 5310B, as shown in FIG.53D, may be used to open the passive valve, allowing for the reagentsfrom an input channel to reach the flow cell.

For example, a dATP reagent may commence to flow from a dATP inputchannel 5390A, around both sides of a liquid loop channel 5305, and intothe input channel for a flow cell 5395. Then, the pressure applied tothe dATP reagent channel 5390A may be reduced or eliminated such thatthe fluid is separated at the location of the passive valve 5380. Abuffer wash cycle may then commence where pressure can be applied to abuffer input channel 5390B such that the fluid is able to cross over thearea of the passive valve 5380, into the liquid loop channel 5305, andfinally into the flow cell input channel 5395. Then, the pressureapplied to the buffer channel 5390B may be reduced or eliminated suchthat the fluid is separated at the location of the passive valve 5380.This process may be continued with a dTTP input channel 5390T, a dCTPinput channel 5390C, and a dGTP input channel 5390G, wherein the inputof nucleotides is alternated with the input of buffer in order to washthe liquid loop channel 5305, the flow cell input channel 5395, and theflow cell in between cycles of different reagents.

Wall Support

Amplification and/or sequencing arrays (e.g., chips) may have one ormore large chambers where the sequencing/amplification array can belocated. In some cases, structural fidelity may become an issue whenthere is a relatively large reaction chamber. For example, the “ceiling”above an array may begin to sag inward proximate to the midpoint betweensidewalls of a chamber. Thus, structural reinforcements may be used inorder to promote improved structural integrity, which may result in amore durable and long-lasting device. Structural reinforcements may be,for example, wall supports placed in one or more locations within areaction chamber such that the weight of the chamber ceiling is moreuniformly distributed.

Isothermal and Solid Phase Amplification Methods

Isothermal Amplification Methods

Polynucleotide amplification is often used for generating large amountsof nucleic acid samples for robust sequencing measurement, and inparticular, sequencing by synthesis. Present embodiments provide systemsand methods applied in polynucleotide amplification. Some examples ofpolynucleotides that may be amplified according to the systems andmethods include DNA, cDNA, modified DNA, synthetic DNA, RNA, mRNA,modified RNA, synthetic RNA, etc. In some embodiments, thepolynucleotide may be single stranded or double stranded.

Some nucleic acids may need to undergo prior treatment via a suitableprotocol before amplification methods described herein may be completed.In one embodiment, for example, mRNA or total RNA may be need to bereversed transcripted to cDNA with reverse transcriptase, likeSuperscriptase, and with a primer containing Poly A or random hexamertagged with nick restriction enzyme sites, a,b,c. The RNA template canbe digested by RNAse H, or base and the generated single stranded,complementary (cDNA) can be ready as a template for amplification. Insome cases, an appropriate primer for the cDNA template can be a randomprimer or a degenerate primer.

In some embodiments, an isothermal amplification reaction may beachieved by use of a first nucleotide primer that comprises a specific,or predetermined, sequence. In other embodiments, the first primer maycomprise a randomized sequence. In a further embodiment, the firstprimer may be a degenerated primer. In some embodiments, the firstprimer may be an oligonucleotide or an oligonucleotide analog. Aplurality of first primers having the same sequence may be used. In thealternative, the use of a plurality of first primers having sequencesthat differ from each other is also contemplated. In another embodiment,the first primers may have the same or similar annealing temperature andmay not have any or minimal complementarities between them.

In some embodiments, it may be desirable to have a sufficiently highconcentration of first primers such that amplification reactionefficiency may be optimized. In a further embodiment, it can bedesirable to select first primers that are less likely to produceprimer-dimer amplification reactions. The first primer can be attachedto a substrate, such as for example, a surface such as a microsensor orglass slide, or a carrier such as a microparticle or a bead. In someembodiments, the carrier may be a magnetic bead ranging in size, forexample, of 20 μm or less, 5 μm or less, 500 nm or less, or 50 nm orless, etc.

In other embodiments, the substrate may have a flat surface, a poroussurface, a crystalline surface, etc. In some embodiments the substratemay be a carrier that is a solid carrier, a porous carrier, a quantumdot, etc. In further embodiments, the substrate can have any shape suchas spherical, flat, rectangular, crystalline, irregular, wells, etc. Insome embodiments, the substrate material may comprise, for example,silicon, silicon-based material, glass, modified or functionalizedglass, magnetic material, plastic, metal, ceramic, gels, acrylic resins,biological material, etc.

In some embodiments, the first primer may be attached to the substratethrough any suitable attachment method. Some exemplary attachmentmethods include DNA hybridization, biotin streptavidin binding, thiolbinding, photo-activated binding, covalent binding, antibody-antigen,physical confinement via hydrogels or other porous polymers, etc., or acombination of methods. In some embodiments, more than one type ofprimer may be attached to the same or different types of substrates.

In one embodiment, the first primer may be, for example, 5, 10, 20, 30,40, 50, 60, 70, etc. base pairs long and hybridize to a desired targetsequence. In some embodiments, it may be preferable to select primersthat have low self-complementarity and high stability in the desiredtemperature or pH range of the amplification reaction.

In some embodiments, wherein the substrate comprises, for example, abead, the bead may be prepared according to the attachment methodsdescribed above such that there is more than one copy of the firstprimer attached to the bead. The concentration of the first primers maydepend on the reagents used and the nature of the specific primersselected. In some embodiments, for example, the concentration of firstprimers on a substrate, such as a magnetic bead, may be 1,000, 10,000,50,000, 100,000, 200,000, 500,000, 1 million, 5 million, 10 million, 50million, etc. first primers per bead, or another concentration whereinthe primers may have the same or different sequences. Each bead may beattached to first primers having the same or different sequences fromthe primers attached to other beads. In a further embodiment,bead-primer complexes can be arranged in amplification arrays thatcontain, for example, 1,000, 10,000, 100,000, 500,000, 1 million, 10million, 500 million, 1 billion, etc. primer-bound beads.

In an alternative embodiment, a combination of primer-bound substrates,such as for example both beads and planar microsensors, may be used inan amplification array.

In some embodiments, at least some portion of the first primer may becomplementary to a DNA template used in an isothermal amplificationreaction. Other reagents may be used for DNA amplification that include,for example, buffers, deoxyribonucleotide triphosphates (dNTPs), ions(e.g., Mg2+), co-factors, primers, polymerase, betain, DMSO, etc.

In order to allow for target-specific hybridization of the primer andtemplate nucleic acid and to prevent non-specific hybridization withother nucleic acids, reaction conditions may be optimized according tosome embodiments. Non-specific hybridization may be reduced by usingstringent reaction conditions or by denature at higher temperature andramp down to the melting temperature (Tm) of the primer. The use ofstringent reaction conditions can help avoid the generation of unwantedreactions. In some embodiments, stringency may be increased by theaddition of organic co-solvents such as, for example, 1-methyl2-pyrrolidinone, formamide, DMSO, polyethyleneimine, polyethyleneglycol, etc.

In some embodiments, temperature ranges such as, for example, 20-95° C.can be used for the isothermal DNA amplification reaction. The desiredtemperature may depend on the type of reagents, such as enzymes, thatare used.

In some embodiments, as shown in FIG. 11, a first primer may be attachedto a carrier, such as for example a bead, in a 5′ to 3′ direction. In analternative embodiment, the first primer may be attached to the beadsuch that one or both ends of the first primer are free and exposed tosolution. The 5′ end of the first primer may be attached to the bead byany suitable method, such as, for example covalent means. Apolynucleotide template with a sequence in the 3′ end that has at leastsome portion complementary to the first primer, such as a DNA template,may be added.

The template nucleic acid used for the amplification reaction may beDNA, RNA, PNA, LNA, a DNA-RNA hybrid, etc. The nucleic acid selected foramplification may be selected from a broad range of sizes or lengths,for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500,700, 1000, 2000, 5,000, 10,000, etc. base pairs. The nucleic acid may besingle stranded or double stranded. In some embodiments, a singlestranded DNA template may be used as the template nucleic acid for DNAamplification. The nucleic acid template can be acquired from anyvirtually any source that contains nucleic acids, such as for examplefrom bacteria, human or animal tissue, plant tissue, fluids such asblood, food, environmental samples, etc.

In some embodiments, as shown in FIG. 12, the DNA template may contain asequence (“Adaptor A”) at the 5′ end that may contain one or more nickrestriction enzyme sites. This adaptor sequence may be, for example, 5,10, 20, 30, 40, 50, 100, etc. base pairs long. Nick restriction enzymes,also called nicking endonucleases, can be utilized. Nickingendonucleases recognize specific sites on the template DNA and generatea nick on the template DNA strand on or near the recognition site. Thenick is generated in the phosphodiester backbone on or near therecognition site. In one embodiment, these enzymes only nick one strandof a double stranded DNA molecule. Examples of nicking endonucleasesthat may be used include, but are not limited to: N.BstNBI, Nt.CviPII,Nt.AlwI, Nt.BspQI, Nb.BsmI, etc. or a combination of nickingendonucleases. The nicking endonuclease, in some embodiments, may beselected to be thermostable. In other embodiments, DNAzymes or Ribozymesmay be used to nick DNA.

In some embodiments, a sequence independent method to introduce nicks orgaps into a DNA strand may involve amplification of template DNA withprimers, one of which contains one or more modified nucleotides, such asfor example dU. In an alternative embodiment, an adaptor containing oneor more dU nucleotides can be ligated to the 5′ end of template DNA.Upon full extension of the first primer, until the 5′ end of thetemplate DNA strand that contains modified nucleotides, the lattermodified nucleotides can be excised by any of commercially availableN-glycosylases/AP-lyases. Thus, the resulting single-stranded gaps mayserve as binding sites for strand displacing DNA polymerase. One exampleof a commercially available product that can be used for this purpose isUSER Enzyme (mixture of Uracil DNA glycosylase and Endonuclease VIII)and it can be obtained from New England BioLabs (NEB). Uracil DNAglycosylase catalyses the excision of uracil base, forming an abasic(apyrimidinic) site while leaving the phosphodiester backbone intact.The lyase activity of Endonuclease VIII breaks the phosphodiesterbackbone at the 3′ and 5′ sides of the abasic site so that base-freedeoxyribose is released. Although the remaining 3′ phosphate may be ahindrance for extension by DNA polymerase, it can be removed byincluding E. coli Endonuclease IV (NEB) into the mixture.

In some embodiments, The dU gapping site can be introduced into theclonal double-stranded DNA by ligation of either nicking or dU siteadaptors.

The DNA template may anneal to the first primer in the orientation thatenables first primer extension leading to the synthesis of a DNA strandcomplementary to the template DNA, as shown in FIG. 12. In someembodiments, the DNA template may anneal to the first primer in a 3′ to5′ direction and is amplified by contacting the DNA template-firstprimer complex with a DNA polymerase and dNTPs. A polymerase is anenzyme that catalyzes the extension and formation of a complementarypolynucleotide based on a template nucleotide. For example, DNApolymerase incorporates dNTPs to allow for the synthesis of a DNA strandthat is complementary to the DNA template strand, starting from thefirst primer region. DNA polymerase moves in a 3′ to 5′ direction alongthe template strand, and synthesizes the complementary DNA strand in a5′ to 3′ direction.

In some embodiments, it may be desirable to select a DNA polymerase thathas no, or limited, exonuclease activity, either in the 3′ to 5′ or the5′ to 3′ direction. In other embodiments, it may be desirable to choosea polymerase that has exonuclease activity to allow for “proofreading”of the growing complementary DNA strand. Since exonuclease activity maydepend on ionic concentration, pH, temperature, buffer, monovalent ioniccomposition, divalent ionic composition, trivalent ionic composition,concentration or presence of dNTPs, etc. these factors can be optimizedto obtain the desired level of exonuclease activity.

Some examples of DNA polymerase that may be used according to someembodiments include, but are not limited to, Klenow DNA polymerase, TaqDNA polymerase, T4 DNA polymerase, VENT DNA polymerase, T7 DNApolymerase, Bst DNA polymerase, Bsu DNA polymerase, etc. or acombination of different DNA polymerases. In a further embodiment, theDNA polymerase may be selected to be thermostable.

In one embodiment, the DNA polymerase (P) can extend the first primer tothe 5′ end of the template DNA in order to form double stranded DNA, asshown in FIG. 13. The nick restriction enzyme sites of Adaptor A in thetemplate DNA strand may also form in the double stranded DNA once DNApolymerase has extended the Adaptor A portion of the template DNAstrand.

In some embodiments, a nick may be introduced into the Adaptor A regionof the template DNA strand by utilizing one or more nick restriction(NR) enzymes, as shown in FIG. 14. In a further embodiment, stranddisplacement DNA polymerase may be added to bind to the nicked site onthe template DNA and perform strand displacing polymerization, as shownin FIG. 15. This may result in the release of the newly-generated singlestranded DNA.

Some examples of suitable strand displacement polymerases according tosome embodiments include VENT DNA polymerase, Phi29, Klenow fragmentpolymerase, T4 DNA polymerase, Bst polymerase, etc. Any one of thepolymerases discussed above or elsewhere herein may be used.

In some embodiments, it may be desirable to select a strand displacingDNA polymerase that lacks 5′ to 3′ exonuclease activity or 3′ to 5′exonuclease activity, in order to help avoid degradation of the DNAstrand being displaced or degradation of the newly synthesized DNAstrand, respectively. In other embodiments, selecting a stranddisplacing DNA polymerase with exonuclease activity may be desirable. Ina further embodiment, a thermostable strand displacing polymerase may beselected.

In another embodiment, a strand displacement factor may be used toenhance strand displacement activity of the polymerase. Some examples ofstrand displacement factors and the corresponding polymerase that theyinteract with include, but are not limited to: E. coli SSB (DNApolymerase II), gp32 protein of T4 bacteriophage (Polymerase gp43), andgp2.5 encoded by T7 bacteriophage (T7 DNA polymerase), recA, betaine,etc.

In a further embodiment, a strand displacing enzyme may be used toperform the strand displacing step and then a polymerase may be added toperform the extension step. Some examples of strand displacing enzymesinclude, but are not limited to, helicase, mismatch repair enzymes (thathave strand displacement capabilities), or modified enzymes (that havestrand displacement capabilities), etc.

In some embodiments, it may be advantageous to select a DNA polymerasethat has high processivity in order to enhance the speed, length, andefficacy of the amplification reaction. In a further embodiment, theprocessivity of the DNA polymerase may be increased by the addition ofprocessivity factors, such as for example, E. coli thioredoxin (for usewith T7 polymerase). Other processivity factors include, for example,sliding clamp proteins such as Archaeal PCNA—Proliferating Cell NuclearAntigen associated with archaebacterial DNA polymerase ∈, bacteriophageT4 gp45 protein associated with T4 DNA polymerase, β subunit of E. coliDNA polymerase III. In other embodiments, protein mediated correctionenzymes may be utilized to improve the fidelity of the DNA polymerase.Protein mediated correction enzymes, such as for example MutS, may beused, or any other suitable enzyme can be used alone or in combination.

In some embodiments, the released single stranded DNA is then free tobind to another first primer at another location on the same or otherbead, and the cycle may be started anew, as shown in FIG. 17. In furtherembodiments, a plurality of first primers, situated in various locationson the bead, may be extended by DNA polymerase to form double strandedDNA with the 5′ end blunted, as shown in FIG. 18. Since the nickingrestriction enzyme site can be restored when new double stranded DNAforms, the nicking, extension, and displacement may be repeated formultiple rounds in order to generate multiple copies of single strandedDNA wherein the sequence of the amplified single stranded DNA is thesame as that of the original DNA template, as shown in FIG. 16.

In some embodiments, the Adaptor A sequence may be designed such that itcontains more than one restriction site. The Adaptor A sequence mayhave, for example, 2, 3, 4, 5, 10, etc. restriction sites. In theembodiment shown in FIG. 12, Adaptor A may contain 3 restriction sites(A, B, and C). If the Adaptor A sequence contains more than onerestriction site, whatever nick restriction site that may remain on thenewly formed double stranded DNA will be recognized by its correspondingnick restriction enzyme. This can create a new nick site on the newlyformed double stranded DNA. This step results in rounds of nicking,extension, and displacement of the DNA, repeated until all orsubstantially all of the first primers on the substrate may be extended.FIG. 19 shows one embodiment of the result after a number of cycles ofamplification with a nicking step. In the exemplary embodiment, theoriginal template DNA had restriction sites A, B, and C and theamplified strands contain either A and B restriction sites, or just siteA.

In some embodiments, isothermal amplification may be completed over anumber of cycles using the method described above. Optionally, in someembodiments, there are additional steps that may be taken, describedbelow, depending on individual needs and requirements.

In a further embodiment, a short, double stranded DNA sequence (AdaptorB), may be used. This adaptor sequence may be, for example, 10, 20, 30,40, 50, 100, etc. base pairs long. Adaptor B may contain one or morenick restriction enzyme sites, as shown in FIG. 20. Adaptor B can ligateto the 5′ end of the double stranded DNA formed by the method describedabove. The steps of nicking, extension, and displacement of DNA may berepeated until all of the first primer on the substrate may be extended,as shown in FIG. 21.

In some embodiments, Adaptor B can be ligated to the 5′ end of thedouble stranded DNA through suitable ligation methods. This may beaccomplished by use of enzymes such as T4 DNA ligase, T3 DNA ligase, E.coli ligase, T7 DNA ligase, Taq DNA ligase, etc. Blunt end ligation maybe enhanced by the addition of compounds, such as for example, PEG 6000,PEG 8000 etc.

In the amplification methods and exemplary embodiments described above,the amplification reaction may be isothermal. Unlike traditionalamplification methods, such as PCR, no temperature cycling is required.

In another embodiment, the isothermal amplification methods describedabove may optionally be followed by an amplification method thatcomprises a nucleic acid denaturation step. Once double stranded DNA isformed using the above methods, the ends of the double stranded DNA thatare not bound to the substrate may be denatured and opened up.

The ease with which double stranded DNA may be separated is representedby its melting temperature. The lower the melting temperature, theeasier the double stranded DNA may be “unzipped”. Double stranded DNAmay be opened by denaturing due to heat or, in some embodiments, by “DNAbreathing”. In some cases, DNA base pairs can stay closed on the orderof a few milliseconds. The localized fluctuations of DNA base pairsopening and closing may be referred to as “DNA breathing” and it isspontaneous, depending in part on thermal fluctuations.

In some embodiments, the opening of the double stranded DNA may allowfor a second primer containing a sequence complementary to the 3′ end ofthe double stranded DNA away from the substrate and with or without nickrestriction enzyme sites to hybridize to that end, as shown in FIG. 22.In other embodiments, an Adaptor C that is partially single stranded andpartially double stranded may be used in the same fashion, as shown inFIG. 22. Alternative embodiments are listed below and may be used aloneor in combination.

In one embodiment, a second primer containing a sequence complementaryto the 3′ end of the DNA may be hybridized to that end. If the secondprimer does not contain any nick restriction enzyme sites, a stranddisplacement DNA polymerase may be used to bind to the 3′ end of thesecond primer and can perform strand displacing polymerization, as shownin FIGS. 23 A-F. This may result in the release of single stranded DNA.This method may be repeated until all or substantially all of the firstprimers on the bead may be extended. In another embodiment, the secondprimer may contain one or more nick restriction enzyme sites. The secondprimer may hybridize to the complementary DNA strand and a nickrestriction enzyme site may form in the double stranded DNA. Nickrestriction enzymes can then be added in order to release the singlestranded DNA. This method may be repeated until all or substantially allof the first primers on the bead may be extended.

In a further embodiment, if using Adaptor C, the nick between Adaptor Cand the double stranded DNA may be repaired by ligase or DNA polymerase.All or substantially all of the first primers on the bead may beextended either by strand displacement DNA polymerase alone (Adaptor Cwithout a nick restriction site) or along with a nick restriction enzyme(Adaptor C with a nick restriction enzyme site),

The denaturing step may be achieved by either the application of heat orthrough heat plus chemical means. In certain embodiments, the nucleicacid may be denatured by a temperature cycle of, for example, 50-60° C.

In a further embodiment, chemical means of denaturing the nucleic acidmay be utilized in addition to heat. For example, NaOH may be applied tothe reaction area in order to denature the nucleic acid. Other chemicalmeans of denaturing the nucleic acid include, but are not limited to:Formamide, Urea, Betain, DMSO, etc.

In some embodiments clonal amplification of the target DNA may beachieved by isothermal transcription-mediated amplification.Single-stranded target DNA can be flanked by two different adaptersequences (A and B). The 5′-end adapter B may contain a unique sequenceon its 3′-side and sequence of the upper strand of the T7 promoter onits 5′-side. The 3′-end adapter A may be complementary to the primer A′,which can be attached to a bead at its 5′-end. The target may attach tothe bead through hybridization between adapter A and the A′ primer. The3′-end of the primer can be extended by Reverse Transcriptase (RT) up tothe 5′-end of the B adapter on target DNA. That may create a doublestranded promoter for T7 RNA Polymerase (T7 RNAP) at the distant end ofthe target DNA. T7 RNAP can initiate transcription of that promoter,synthesizing hundreds of RNA transcripts comprising target DNA sequencesflanked by sequences of adapter A and unique part of adapter B. The RNAtranscripts discussed above may hybridize to other A′ primers on thesame bead. In some embodiments, those primers can be extended by RT upto the 5′-end of RNA transcripts, thus creating DNA:RNA heteroduplexes.

In one embodiment, the RNA strand of the duplexes discussed above may behydrolyzed by either RNase H activity of the RT or by the addition ofRNase H enzyme. The second primer (T7 primer) comprising of the sequenceidentical to the B adapter hybridizes to the cDNA strand. The RT maycontinue to extend the cDNA strand up to the 5′-end of T7 primer. The T7primer may be blocked at its 3′-end and may not be extended. At thispoint the double-stranded T7 promoter may be created at the end of thetarget that is distant from the bead. T7 RNAP may synthesize hundreds ofRNA transcripts off the template described above. Those transcripts mayhybridize to other A′ primers on the same bead thus initiating anothercycle of repetitive cDNA and RNA synthesis. The process can continueuntil all primers on the bead are extended and turn into single-strandedDNA molecules that may be attached to the bead and can comprise of thesequence complimentary to the original DNA target flanked by adapters.The adapter sequence at the 3′-end of the above molecules may becomplimentary to the adapter B and can be used for hybridization of asequencing primer.

In one embodiment, the amplification methods described above may becarried out in a reaction chamber, a well, a virtual well, an array,etc. The amplification methods may be used in conjunction with anintegrated system. For example, the integrated system may be anintegrated sequencing platform and may include a DNA extraction system,a library construction system, an amplification system an enrichmentsystem, and a sequencing system. The integrated sequencing platform caninclude all of these systems within a singlemicrofluidic/microelectronic device (or “chip”).

Various types of amplification protocols are contemplated by this methodsuch as, for example, isothermal amplification, rolling circleamplification, strand-displacement amplification (SDA), self-sustainingsequence replication (3SR), bridge amplification, nucleic acidsequence-based amplification (NASBA), polymerase chain reaction,transcription-mediated amplification (TMA), ligase chain reaction (LCR),etc. or a combination of amplification protocols.

The amplification methods described above may also be used foramplification applications wherein clonally amplification is notdesired. For example, the methods may be used for creating ampliconsfrom different sample populations. Methods for keeping samples separatemay be used, such as for example, DNA barcoding or using carriers ofdifferent sizes/colors. In some embodiments, the amplification methodsdescribed above may be used for amplification of different samplepopulations before using a DNA hybridization array to determine thepresence of a gene of interest.

Solid Phase Amplification

In some embodiments, as shown in examples of FIGS. 68A, 68B, and 68C, anamplification reaction may be a solid phase amplification reaction,using primers configured in a variety of fashions. In some embodiments,as shown in FIG. 68A, a first primer 6800 may be on a surface, such asthe surface of a bead 6850, and a second primer 6810 may be in solution6830. In other embodiments as shown in FIG. 68B, primers 6800 & 6810 maybe on the bead 6850. In other embodiments, as shown in FIG. 68C, primers6800 & 6810 may be present in solution 6830. In some embodiments, oneprimer of 6800 and 6810 or both primers may be also present on the bead6850. In a further embodiment, the amplification may be performedwhereby one primer of 6800 & 6810 is present in solution 6830, and oneprimer or both primers are also present on the bead 6850.

Joule Heating for Improved Isolation and/or Concentration of Species andControl Via Heat Cycling

Joule Heating for Improved Concentration of Species

As described elsewhere herein, species such as reagents suitable fornucleic acid amplification and sample nucleic acid can be concentratedat pixels of array via the generation of electric fields. Examples ofsuch concentrating are also described in PCT Patent Application No.PCT/US2011/054769, PCT Patent Application No. PCT/US2012/039880, PCTPatent Application No. PCT/US2012/067645, and U.S. patent applicationSer. No. 13/481,858, which applications are incorporated herein byreference in their entireties.

In some embodiments, electric fields can be used to attract templatenucleic acids (e.g., template DNA), dNTPs, and primers to a “confinementcell” region or “chamber-free amplification” region. In some cases, apixel of an array may comprise such a confinement cell region orchamber-free amplification region. In some cases, each pixel of an arraymay comprise its own confinement cell region or chamber-freeamplification region. Following the attraction of reagents and templatenucleic acid, amplification of the template can begin in regions of eachcell where template nucleic acid is located. During amplification, theelectric fields may aid in preventing cross contamination betweendifferent confinement cells undergoing amplification by retainingamplicons. In order to insure that polyclonal regions are not generated,the concentration of input nucleic acid (e.g., DNA) may need to be lowenough such that most confinement cells have one or zero sample DNAmolecules. Nucleic acid samples can be single stranded or doublestranded depending on the amplification methodology. Moreover, asdescribed elsewhere herein, sample nucleic acid may be associate with acarrier, such as, for example, a bead. In some embodiments, samplenucleic acid molecules may be added to carriers (e.g., beads) prior toor after loading of the carriers into an amplification array. In somecases, electric fields may also be useful in concentrating reagents andother species in sequencing reactions.

Some factors associated with amplification methods that include theconfinement of reagents at pixels of an array via an electric field andpotentially subject to optimization include the frequency, voltage, typeof signal input, shape of signal input, absolute value of voltage, dutycycle, and dimensions of the electric field confinement cell used toconfine sample nucleic acid (e.g., template DNA) and reagents such as apolymerase and generated amplicons. If confinement were the onlyconsideration, it may be possible to confine almost any size ofamplicon, including amplicons of fairly small size. An electric fieldthat is strong enough to ensure proper confinement, however, may alsoprevent proper activity of other reagents, such as, for example, apolymerase, during an amplification reaction (e.g., PCR, isothermalamplification, primer extension, etc.). For example, a strong electricfield may prevent a polymerase from binding with a template nucleic acidand/or may exert a force on a polymerase that dissociates it from atemplate nucleic acid and extended primer. In another example, a strongelectric field may exert a force on an extended primer that dissociatesit from a template nucleic. Proper arrangement and operating conditions(e.g., applied voltage, frequency, duty cycle, reaction conditions,etc.) of an electric field may help to ensure that the electric fielddoes not pull the polymerase and/or extended primer from a complex ofthe template nucleic acid (e.g., template DNA), extended primer, andpolymerase.

In some embodiments, it may be desirable to optimize a combination offrequency, voltage, and size of a confinement cell, depending on thesize of an amplicon generated in the confinement cell. For example, thesize of a confinement cell can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreμm in length or diameter and of various shapes that include, forexample, squares, rectangles, circles, hexagons, etc. or any othershape. In some embodiments, the frequency can range from a DC signal (0Hz) to an AC signal of a few Hz to several kHz or MHz. In someembodiments, the voltage can consist of, for example, 0.5 V or 1V ACwith 500 Hz frequency with a 0.8V or 1.2 V DC offset.

Amplification within a confinement cell can be achieved using eitherelectrophoresis or a dielectrophoretic field, or both. In order toinduce dielectrophoresis, an array of electrodes can be used to createnon-uniform electric fields. The electrode configuration may takevarious forms, including an outer electrode that defines the outside ofthe confinement cell and an inner electrode, or there may be two innerelectrodes proximate to a carrier, for example, a bead (e.g., a magneticbead) with a magnet located such that it retains the carrier proximateto the electrodes.

In some embodiments, inner electrodes may have alternating positive andnegative polarities or charges so as to concentrate template nucleicacid and reagents in close proximity to a carrier (e.g., bead) locatedbetween the inner electrodes. The inner electrodes can alternate backand forth between positive and negative charges. In this manner,template nucleic acid and reagents used for amplification may be passedback and forth between the inner electrodes, concentrated in an area onor proximate to the carrier, and also prevented from attaching to,passing by, or getting in close proximity to an electrode of oppositecharge (outside electrode). In some cases, an outer electrode isnegatively charged. The electric field generated by a negatively chargedouter electrode can be an additional barrier, preventing the crossingand diffusion of negatively charged nucleic acid (e.g., DNA) away fromthe confinement cell. In this manner, template nucleic acid (e.g., DNA)and reagents may be concentrated on or near the carrier, which can allowfor an increase in efficiency of the amplification reaction. In someembodiments, the impact of local pH change around a carrier may becontrolled with the distance of the inner electrodes from the carrier orwith a coating layer such as a bleach-type material (e.g., HQ), apolymer, or another coating material.

In some cases, when using a dielectrophoretic field, flow may begenerated by dielectrophoresis. FIG. 61A illustrates an example ofdielectrophoresis-induced flow (or electroosmotic flow) 6100 aboveelectrodes 6120 of a pixel. In some cases, the retaining of amplicons,reagents, and/or template nucleic acid, etc. in a confinement cell ofthe pixel may become problematic, as the flow 6100 can cause suchspecies to drift to another area. If transport of the species viaelectroosmotic flow is sufficient, there may be contamination of otherpixels, due to amplicons and/or other species such as template nucleicacid moving from a pixel to a neighboring pixel via transport.Furthermore, in the case of clonal amplification, the electroosmoticflow may negatively affect amplification efficiency if it interfereswith amplicons binding to a carrier.

In some cases, Joule heating may be used to help address potentialcross-contamination issues that may arise from electroosmotic transportof species. Joule heating generally refers to heat that can be generateddue to electric current passing through a conductor, such as anelectrode. Such heat can create a counter flow, in some cases, suitableto offset the flow that can be generated due to dielectrophoresis. Jouleheating can be described by the following equation:Q=I²Rtwhere Q is heat (e.g., heat in Joules), I is electrical current (e.g.,current in amps), R is electrical resistance (e.g., electricalresistance in ohms), and t is time (e.g., time in seconds).

In some embodiments, Joule heating may be used to generate movement inan opposite or other direction that can counter flow generated bydielectrophoresis. In some cases, such flow may cancel out flow fromdielectrophoresis, an example of which is shown in FIG. 61B. Jouleheating-induced flow 6110 moving in the opposite direction of thedielectrophoresis-induced flow 6100 may result in a flow that leads tothe isolation and/or concentration of species, such as, for examplenucleotides, polymerase, nucleic acids (e.g., DNA), other reagents,other charged species, etc. in and around area 6130, proximate to theelectrodes 6120. In some cases, the flow may be circular, turbulent,laminar, or any other type of flow.

In some embodiments, there may be only one electrode per pixel. In otherembodiments, as shown in the examples of FIGS. 61A-B, there may be onemiddle electrode and two electrodes proximate to the middle electrode ina pixel. Other configurations of 3 or more, 4 or more, 5 or more, etc.electrodes can be used depending on the level of confinement and thespecific application. Joule heating may be used with such configurationsor any other suitable configuration.

In some embodiments, electrophoresis may be combined withdielectrophoresis and Joule heating. A DC current may be generated byone or more outer electrodes in order to help contain the amplicons.There may be one or more inner electrodes that operate using DC currentor AC current (electrophoresis or dielectrophoresis, respectively). Insome embodiments, Joule heating-induced flow may be adjusted such thatit does not cancel out the dielectrophoresis-induced flow. In someembodiments, dielectrophoresis-induced flow is permitted to create aflow, with or without using Joule heating.

In some cases, a flow around electrodes of a pixel may be desirable forapplications, such as, for example, washing of reagents, electrodes,carriers, and/or an array. Such flow may be circular, turbulent,laminar, or any other type of flow. In some cases, flow aroundelectrodes of a pixel may be used to mix or wash or may aid in washingthe reagents off the array. In some embodiments, flow generated by Jouleheating may be used to optimize reagent delivery. For example, flowgenerated by Joule heating may aid in distributing reagents and/ortemplate nucleic acid across the array more efficiently than other formsof reagent delivery. Improvements in the efficiency of species deliverymay lead to a decrease in delivery time and may shorten the time neededto conduct biological processes of interest, such as, for example,nucleic acid (e.g., DNA) amplification, or nucleic acid (e.g., DNA)sequencing.

Control Via Heat Cycling

When inner and/or outer electrodes use a DC current for electrophoreticconcentration or confinement of amplicons, electrolysis may occur athigher voltages and may cause issues such as, for example, bubblesand/or a drop in pH in regions proximate to an electrode. In someembodiments the pH change can be reduced or eliminated by using a higherbuffer concentration. In some embodiments, the generation of bubbles canbe reduced or eliminated by coating the electrodes with a suitablematerial such as, for example, hydroquinone (HQ), or another type ofcoating layer. In some cases, the generation of bubbles can be reducedor eliminated by using electrodes with a larger surface area exposed toa liquid and/or by using porous electrodes. Examples of suitable porouselectrodes include Black Platinum electrodes and Iridium electrodes.

In generating a DC field for electrophoretic concentration orconfinement, electrolysis products can build up, with non-limitingexamples of such electrolysis products that include hydronium andhydroxide ions. To minimize effects from these ions, the DC field can bepulsed so that the net DC effect is much lower. In some embodiments apulsed duty cycle can be reduced after species (e.g., reagents, nucleicacid) have migrated closer to an electrode. In some embodiments, a DCfield can be used to concentrate species and an AC field can be used tomaintain concentration and/or confinement of electrolysis products. Inother embodiments, both a DC field and an AC field can be used forconcentration and confinement.

In generating a DC pulse, voltage can be raised to a higher voltage fora period of time, and then be reduced to zero volts (or a lower voltage)with no or minimum electrophoresis for a period of time. The lower orzero volt time period can allow for the diffusion of any bubbles thatmay have formed as a result of electrolysis.

Although a pulsed DC field may be used to counter the negative effectsof electrolysis, such as bubble generation or a pH change due togeneration of byproducts, the pulsed signal may increase the likelihoodthat amplicons or other species are able to drift away from aconfinement cell during the period in which the DC pulse is at a lowvoltage. In some embodiments, heat cycling during amplification can beused or synchronized with a DC pulse so that when the DC pulse is at alow voltage, a solution within the confinement cell is at a lowtemperature, and when the DC pulse is at a high voltage, a solutionwithin the confinement cell is at a higher temperature, as example ofwhich is shown in FIG. 62A. As shown in the example of FIG. 62A, whenthe DC pulse is at a higher voltage 6250, the temperature may also beset to a higher level 6245. During this time period 6200, as shown inFIG. 62B, nucleic acid (e.g., DNA) subject to amplification in aconfinement cell may be a combination of double and single strandednucleic acid. In some cases, a majority of the nucleic acid can besingle stranded and some nucleic acid can be in solution. When thevoltage is at a higher level 6250, there can be a reduced chance ofnucleic acid in solution within a confinement cell migrating intoanother confinement cell.

During the second time period 6205, however, when the voltage is reduced6235, the temperature may also be reduced 6230. In some cases, duringsuch a time period, the number of double-stranded nucleic acids bound toa carrier (e.g., bead) within a confinement cell can be increased, suchthat most of the nucleic acid is bound to the carrier. Binding can helpreduce migration of amplicons from the confinement cell.

In some embodiments, a DC field may be applied in a pulsed fashion inorder to prevent heating of the electrodes. Excessive heat generated bythe electrodes may have a negative impact on a biochemical reactions ofinterest, such as for example nucleotide incorporation, amplification,sequencing, etc. Pulsing of a DC field may provide time for an electrodeto cool down between pulses.

In some embodiments, the voltage of a DC and/or AC electric field may beadjusted such that it promotes dielectrophoretically-induced flow,electroosmotic flow, or other similar effect. In some embodiments,voltage, frequency, electrode shape, electrode configuration, etc. mayall be configured in order to promote a specific type of desired flow.In some cases, an electrode array may be patterned such that thearrangement of the electrodes in the array is conducive to creating adesired flow pattern. Such a configuration may be desirable for betterwashing of reagents, carriers, the array, etc. and/or optimized deliveryof reagents. The mixing due to the flow may allow for betterdistribution, and thus faster delivery of reagents. The flow may alsoaid in the removal of reagents from the array.

In some embodiments, a combination of AC and DC applied fields may beused. In some embodiments, Joule heating or heat generation may be usedin applications requiring heat, such as for example, thermocycling, toprovide localized heating and/or temperature cycling.

Library Construction Systems and Nucleic Acid Fragmentation Methods

An integrated system may comprise a library construction system (e.g.,nucleic acid library construction system), which may include afragmentation and/or size selection element. An example of a libraryconstruction system is shown in FIG. 63. As shown in FIG. 63, a libraryconstruction system may include a nucleic acid (e.g., DNA) fragmentationand size selection element 6316. The fragmentation and size selectionelement 6316 can be configured to produce double-stranded nucleic acidfragments, which may or may not have blunted ends, via the elements andmethods described below. The fragmentation and size selection element6316 can include one or more microfluidic channels 6322 within whichnucleic acid may be disposed along with a set of fragmentation beads6324. Nucleic acid 6312 collected in a nucleic acid (e.g., DNA)extraction system (shown for example in FIG. 63) can be conveyed or“injected” into the nucleic acid (e.g., DNA) fragmentation and sizeselection element 6316 by any suitable means (e.g., pressurizedinjection, electrophoretic movement, gravity feed, heat-inducedmovement, ultrasonic movement and/or the like). Similarly, fragmentationbeads 6324 can be conveyed into the nucleic acid (e.g., DNA)fragmentation element and size selection element 6316 by any suitablemeans.

The fragmentation element and/or size selection element 6316 may includea pump 6326 to produce movement of a fluid (e.g., a fluid comprisingnucleic acid (e.g., DNA) and fragmentation beads 6324) within amicrofluidic channel 6322. The pump 6326 can be, for example, aperistaltic pump. In some embodiments, the pump 6326 can include one ormore microfluidic elements in fluid communication with the microfluidicchannel 6322, and may have a flexible side-wall that, when deformed,produces a flow within the microfluidic channel 6322. In otherembodiments, however, any other suitable mechanism can be used as analternative or in addition to produce movement fluid within themicrofluidic channel 6322, with non-limiting examples, that includeselective heating and cooling of the fluid, pneumatic pressurization ofthe microfluidic channel, electrophoretic motion, or the like.

The fragmentation beads 6324 can be constructed from any materialsuitable for separating, cutting and/or otherwise dividing a nucleicacid (e.g., DNA) into nucleic acid fragments (e.g., DNA fragments). Insome embodiments, the fragmentation beads 6324 can be constructed fromglass, polydimethylsiloxane (PDMS), ceramic or the like. Moreover, thefragmentation beads 6324 can have any suitable size and/or geometry suchthat the fragmentation element produces fragments having the desiredcharacteristics (e.g., length, strand characteristics, or the like). Forexample, in some embodiments, the fragmentation beads 6324 can besubstantially spherical and can have a diameter of 50 μm or less. Inother embodiments, the fragmentation beads 6124 can have a diameter of500 nm or less, or any diameter between 50 μm and 500 nm.

Moreover, the size and/or geometry of the microfluidic channel 6322(e.g., cross-sectional shape, aspect ratio or the like) can be selectedsuch that the movement of the nucleic acid (e.g., DNA) within themicrofluidic channel 6322 and contact of the nucleic acid with thefragmentation beads 6324 fragments (e.g., via shearing) the nucleic acidas desired. In some embodiments, the microfluidic channel 6322 may be inthe range of 1 to 500 μm in hydraulic diameter (i.e., thecross-sectional area of the microfluidic channel 6322 can besubstantially rectangular, thus the size can be represented as ahydraulic diameter). In other embodiments, the hydraulic diameter of themicrofluidic channel 6322 can be in the range of 10 to 200 μm. In yetother embodiments, the hydraulic diameter of the microfluidic channel6322 can be in the range of 500 nm or less. In other embodiments, themicrofluidic channel 6322 can have any suitable shape, such assemi-circular, oval, tapered or the like. In some embodiments enzymaticpolishing of sheared nucleic acid (e.g., DNA) ends can be done such thatthe ends are blunt ends.

In other embodiments, an enzymatic solution can be conveyed into themicrofluidic channel 6322 to, at least partially, produce enzymaticfragmentation of nucleic acid (e.g., DNA).

In some embodiments, as shown in an example of FIGS. 64A and 64B,liquids with different flow rates may be used to fragment nucleic acids(e.g., DNA). The flow rate of the first liquid 6400 may be faster orslower than the flow rate of the second liquid 6410. When the nucleicacids, such as for (e.g., DNA) 6420, comes into contact with theinterface 6430 that exists between the two liquids due to thedifferences in flow rate, the resulting shear force on the nucleic acid6420 may result in nucleic acid fragmentation 6440. Moreover, thenucleic acid can be elongated or stretched via the aid of an electricfield. The effect of shear force, electric force, or other forces mayresult in the fragmentation of the nucleic acid.

In some embodiments, illustrated in an example of FIGS. 65A and 65B,nucleic acid (e.g., DNA) 6520 may be fixed to a surface, such as forexample a bead 6550. The bead-bound nucleic acid 6520 may be exposed tofluid flow 6500 from a surrounding bulk solution. Since some part of thenucleic acid 6520 is bound to a fixed point, exposure to flow 6500 mayresult in a shear force on the nucleic acid, which can lead to nucleicacid fragmentation 6540.

In some embodiments, as shown in an example of FIGS. 66A and 66B, eachend of a nucleic acid (e.g., DNA) 6620 may be fixed at each end to acarrier, such as beads 6651 and 6652. One bead 6652 may be coated with apositively charged material 6660. Materials that may be used to create apositively charged coating on the bead 6650 include, for example,materials comprising amines. The other bead 6651 may be coated with amaterial that imparts a negative charge 6665 on the bead 6651. Examplesof suitable materials include, for example, materials comprisingcarboxyl groups. Once each end of the nucleic 6620 is fixed to beads,the bead-nucleic acid structure may be exposed to an electric field6625, the can be generated by one or more electrodes 6680. The electricfield 6625 may induce movement in the beads in opposite directions 6675,due to their respective charge, and the resulting tensional stress onthe nucleic acid 6620 may result in nucleic acid fragmentation 6640. Insome embodiments, one bead can be held in a fixed location and a secondbead may move due to electric fields (electrophoretic ordielectrophoretic force or fluidics). The separated beads can then bedirected to sensors for analysis, such as nucleic acid sequencing, asdescribed elsewhere herein.

In some embodiments, as shown in an example of FIGS. 67A and 67B,nucleic acid (e.g., DNA) 6720 may be passed through a nanochannel 6760.A microchannel 6765 may be located within or proximate to thenanochannel 6760 such that the microchannel 6765 and nanochannel 6760are in fluidic contact. The difference in flow rate of the nanochannelfluid 6710 versus microchannel fluid 6700 may result in a shearing forceon the nucleic acid 6720 such that nucleic acid fragmentation 6740result at the fluidic interface 6790. In some cases, the nucleic acid6720 may be bound to a carrier, such as a bead, or it may be free insolution.

In some embodiments, sonication may be used to fragment nucleic acids.Any suitable sonication method may be used. For example, sonication canbe created by MEMS structures or other structures (e.g., structures withconcentric arcs with different radius). In some embodiments, sonicationcan create microbubbles in a fluid in which the nucleic acid (e.g., DNA)is suspended. Gaseous cavitation that results from the microbubbles cancreate microstreams that may fragment the surrounding nucleic acid.Fragmentation methods described herein may be performed in amicrofluidic channel, in a separate microchamber, on a microchip, etc.

Microfluidic Field-Programmable Gate Array (FPGA) Grid and Modules

The integrated devices described herein provide a customizable platformfor high throughput analysis of biological and chemical reactions ofinterest. In some cases, an integrated may include microfluidictechnology for high throughput analyses. Accordingly, an integratedplatform may comprise one or more integrated microfluidic devices.Methods for configuring such microfluidic devices to suit individualrequirements are provided herein.

In some embodiments, the integrated microfluidic devices may be formedfrom a substrate wherein a plurality of microfluidic channels may beembedded into the substrate.

In some embodiments, the microfluidic channels may be configured to forma grid pattern throughout the substrate or in some portion thereof. Forexample, the microfluidic channels may be arranged as a plurality ofintersecting microfluidic channels along the x, y, and z-axes of thesubstrate. This configuration may allow for a customizable platformwherein the microfluidic channels of the grid may be selectively opened,closed, and/or allowed to intersect with other channels. In anotherembodiment, the microfluidic channels may have valves for controllingflow.

The microfluidic channels may be in fluidic contact with one or moremodules. The module may perform a desired function, for example, as asample preparation module, a nucleic acid (e.g., DNA) amplificationarray module, a nucleic acid (e.g., DNA) sequencing array module, etc.or a combination of functions. The modules may be in fluidic contactwith one or more microfluidic channels via a connection, such as forexample a socket connection, wherein there may be an air-tight andfluid-tight seal at the connection juncture.

Various fluidic “paths” may be created wherein one or more modules maybe interconnected via one or more channel paths. The number and/or typeof input or output microfluidic channels in fluidic contact with themodules may be determined in the same manner.

In some embodiments, the integrated microfluidic devices may be formedfrom a substrate wherein a plurality of microfluidic channels may beembedded into the substrate. The substrate material may be PDMS,Plexiglass, polycarbonate, poly (methyl methacrylate) (PMMA), cyclicolefin copolymer (COC), polyamide, silicon, glass, quartz, etc. oranother material. Depending on the particular application, the substratematerial may be rigid or it may be flexible.

The microfluidic channels may have a cross section that is circular,elliptical, square, rectangular, etc. or another shape. The dimensionsof the microfluidic channel may vary. In some embodiments, themicrofluidic channel may have a diameter of about 100 nm, 500 nm, 1 μm,10 μm, 50 μm, 100 μm, 500 μm, etc.

The microfluidic channels may be configured to form a grid patternthroughout the substrate or in some portion thereof. In someembodiments, for example, the microfluidic channels may be arranged as aplurality of intersecting microfluidic channels along the x, y, andz-axes of the substrate. This configuration may allow for a customizableplatform wherein the microfluidic channels of the grid may beselectively opened, closed, and/or allowed to intersect with otherchannels. FIG. 69 shows a 3D line drawing of the x, y, and z axes forclarification. FIG. 70A shows one embodiment of the top view of themicrofluidic device. FIGS. 70B and 70C show side views of themicrofluidic device, in two different embodiments. FIG. 70B shows adevice with three layers of channels on the x and y axes whereas FIG.70C shows a device with just one layer of channels on the x and y axes.In some embodiments, the microfluidic device may have 1, 2, 3, 4, 5, 10,50, etc. layers of channels on the x and y axes. FIGS. 70D and 70E showside views of a microfluidic device with three layers and one layer,respectively, but with an optional base layer 7010 for support.

In some embodiments, the microfluidic device may be fabricated using aplurality of layers. FIG. 71 illustrates an exploded view top view of anexemplary device with four layers. There may be a base layer 7110,optionally with openings along the z-axis of the substrate (openings notshown). The second layer 7120 may have openings along the z-axis of thesubstrate for channels running along the z-axis 7122 in conjunction withmicrofluidic channel along the x-axis 7124. The third layer 7130 mayhave openings along the z-axis of the substrate for microfluidicchannels running along the z-axis 7132 in addition to microfluidicchannels along the y-axis of the substrate 7134. Finally, there may be atop layer 7140 with openings along the z-axis for the microfluidicchannels running along the z-axis 7142. The openings and channels may bealigned such that they intersect in a grid format, as shown by the topview in FIG. 70A. Thus, the “default” position of this configuration iswith all the channels intersecting and in the “open” position. In otherembodiments, some portion of the substrate may have channels thatintersect while other portions of the substrate may have channels thatdo not intersect.

These microfluidic channels may be in fluidic contact with one or moremodules. The module may perform a desired function, for example, as asample preparation module, a nucleic acid (e.g., DNA) amplificationarray module, a nucleic acid (e.g., DNA) sequencing array module, etc.or a combination of functions. FIG. 72A shows one embodiment of themicrofluidic device 7200 with a sample preparation module 7210 and anucleic acid (e.g., DNA) amplification module 7220.

In a further embodiment, modules may be placed above, within, or belowthe channels of the microfluidic device. FIG. 72B shows a side view ofthe microfluidic device 7200 of FIG. 72A wherein the amplificationmodule 7220 and the sample preparation module 7210 are located on top ofthe microfluidic device 7200, above the microfluidic channels 7230. Onepotential connection path is shown by a dashed line 7250, where themicrofluidic channels along the path are open and in fluidic contactwith the modules. FIG. 72C shows another embodiment of microfluidicdevice 7200, wherein the amplification module 7220 and the samplepreparation module 7210 are embedded within microfluidic device 7200.One possible connection path 7250 is shown.

There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, etc. modules wherein all or some number of the modules areconnected. FIG. 73A shows a microfluidic device 7300 with five modules.There may be, for example, two sample preparation modules 7310, twonucleic acid (e.g., DNA) amplification modules 7320 and a nucleic acid(e.g., DNA) sequencing module 7325. FIG. 73B shows the microfluidicdevice 7300 in a configuration where the modules are located below themicrofluidic channels 7330 and on the bottom of microfluidic device7300. A cover component 7340 for the microfluidic device 7300 in thistype of configuration may be desirable since the modules are “upsidedown”. FIG. 73C shows an exploded view of the microfluidic device 7300and the cover component 7340 for clarification.

The connection path between the modules may vary and depend uponindividual needs. FIGS. 74A and 74B show two exemplary microfluidicdevices 7400 with different connection paths 7450 through themicrofluidic channels 7330 that connect the modules 7440. The modules7440 may be of the same or different types. Since the channels are in an“open” position, once a desired connecting path between the modules isdetermined, the remaining channels not on the path may be closed througha variety of methods. The connecting path may be a straight line betweenone or more points, or it may travel along the x, y, and z axes in anyconfiguration. This type of more convoluted path may be likened to theconcept of Manhattan routing for field-programmable gate array (FPGA)circuits.

Methods for closing the microfluidic channels that are not along theconnection path may depend on the substrate material being used. In someembodiments, the microfluidic channels may be partially or completelyclosed, either at some point along their length or at an intersection oftwo or more channels. Methods for fusing materials may be used to closethe channels, such as for example, lasers, targeted ultrasound, UVlight, etc. In other embodiments, the channels may be closed usingvalves or gates. In another embodiment, the channels may be closed byusing one or more polymers to dissolve the structure in the desired areaof the channel, fusing the area closed.

In some embodiments, the channels may be closed using pins. The pins maybe constructed of metal, plastic, glass, etc. or any other suitablematerial. FIG. 75 shows a microfluidic device 7500 with a samplepreparation module 7510 and a nucleic acid (e.g., DNA) sequencing module7520. The connection path 7550 runs in a straight line along themicrofluidic channels 7530. The solid line 7570 is used to illustratethe area of the channels that should be closed in order to ensure thatthe fluid remains only in the modules and the connection path 7550. Theconnection path 7550 is shown to extend to the ends of microfluidicdevice 7500 (e.g., may be connected to an outside source for reagents),but in other embodiments the connection path 7550 may only be betweenthe modules (e.g., with reagents being applied directly to the module,without using microfluidic channels 7530). A cross sectional, side-view7580 is also shown of a closed area 7570. Pins 7590 are inserted intothe microfluidic channels 7530 and, in this embodiment, run along thez-axis. The pins 7590 serve to close the desired area 7570 in order tohelp ensure there is no leakage into other parts of the microfluidicdevice 7500 from the connection path 7550.

FIG. 76 shows a variety of example pins that may be used to close thechannels. These pins may be inserted along the x, y, and/or z-axes.Their dimensions depend on the dimensions of the microfluidic channelsand the pins generally may be flush with the walls of the microfluidicchannel in order to prevent leakage. FIG. 76A shows a pin 7600A thatcloses some channels, but leaves an opening 7605 for one channel on onelayer. FIG. 76B shows a pin 7600B with an opening 7610 that connects twochannels and two different layers. FIG. 76C shows a cross section of thechannels 7670 that pin 7600B connects, for clarification. FIG. 76Dillustrates a pin 7600D that has an elbow-shaped opening 7615 that canbe used to connect a channel running along the x-axis with a channelrunning along the y-axis. Pin 7600D may be used to close other channelsat levels above and below the opening 7615.

In some embodiments, the substrate may be constructed from a variety ofmaterials. In some embodiments, the channels may be closed using avariety of methods, depending on the physical characteristics of thematerial where the channels are being closed. In one embodiment, if thesubstrate is constructed from a variety of materials, a polymer thatinteracts with less than all of the materials to close the channels inthose materials may be used. For example, as shown in FIG. 77 if thesubstrate of the microfluidic device 7700 is constructed from materials“A”, “B”, “C”, and “D”, a polymer that only has an effect on substrates“A” and “B” may be used in order to close the channels in those regions,leaving the microfluidic channels in regions constructed of materials“C” and “D” open. The boundary lines in FIG. 77 are for illustrationpurposes, as the substrate materials A, B, C, and D may be fusedtogether to form a single structure.

In another embodiment, all or some portion of the microfluidic channelson the x-axis, y-axis, and/or z-axis are configured such that they donot intersect. Accordingly, the microfluidic channels that do notintersect are “closed” in that area in that they are not connected suchthat fluid may pass from one channel to the other.

In some embodiments, the microfluidic device may be fabricated using aplurality of layers. There may be a base layer, optionally with openingsalong the z-axis of the substrate. The next layer may have openingsalong the z-axis of the substrate in conjunction with microfluidicchannels along the x-axis. The following layer may have openings alongthe z-axis of the substrate in addition to microfluidic channels alongthe y-axis of the substrate. Finally, there may be a top layer withopenings along the z-axis. The openings and channels may be aligned suchthat they are in a grid format, but do not intersect. Thus, the“default” position of this configuration is with all the channels in a“closed” position. In other embodiments, some portion of the substratemay have channels that intersect while other portions of the substratemay have channels that do not intersect.

These microfluidic channels may be in fluidic contact with one or moremodules. The module may perform a desired function, for example, as asample preparation module, a nucleic acid (e.g., DNA) amplificationarray module, a nucleic acid (e.g., DNA) sequencing array module, etc.or a combination of functions.

In a further embodiment, modules may be placed above, within, or belowthe microfluidic device. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, etc. modules wherein all or somenumber of the modules are connected. The connection path between themodules may vary and depend upon individual needs. Since the channelsare in an “closed” position, once a desired connecting path between themodules is determined, the channels along the path may be connectedusing a variety of methods. The connecting path may be a straight linebetween one or more points, or it may travel along the x, y, and z axesin any configuration. This type of more convoluted path may be likenedto the concept of Manhattan routing for FPGA circuits.

The methods for connecting the microfluidic channels that are along theconnection path may depend on the substrate material being used. Methodsfor connecting channels in a substrate that do not intersect may beused, such as for example, lasers, polymers, pins etc. FIGS. 76B and 76Cshow how pin 7600B can be used to connect two channels on differentlayers. In other embodiments, the channels may be open using valves orgates.

The modules may be in fluidic contact with one or more microfluidicchannels via a connection, such as for example a socket connection,wherein there may be an air-tight and fluid-tight seal at the connectionjuncture.

Various fluidic “paths” may be created wherein one or more modules maybe interconnected via one or more channel paths. The number and/or typeof input or output microfluidic channels in fluidic contact with themodules may be determined in the same manner.

Integrated Biological Analysis Systems

The devices and methods provided herein are related to reconfigurable,multiplexed autonomous diagnostic platforms that can enable diseaseprevention and facilitate the accurate administration of therapeutics.The integrated point-of-care systems (an example system shown in FIG.78) incorporate automated sample (e.g., biospecimen) collection andpreservation in addition to simultaneous on-chip analysis of one or morevarious analytes, such as, for example, nucleic acids, proteins,antibodies, antigens, cells, and/or other biomolecules of the sample. Asample may be, for example, blood (e.g., whole blood), a culture swab,urine, stool, tissue, or other biological sample and analysis may be,for example, for the purpose of screening for drug-resistant biomarkers.The sample can be obtained from a subject, such as a subject receivingtherapy, having a disease or other health condition, or suspected ofhaving a disease or other heath condition. In some embodiments, suchtechnology can be used to construct an integrated, self-powered,microfluidic biological sample analysis system suitable for low-cost,sample-to-answer point of care (POC) or point of service (POS)diagnostics. An example of a integrated, self-powered, microfluidicbiological sample analysis system can be found in I. K. Dimov et al.,Lab Chip 11, 845, which is incorporated herein by reference in itsentirety.

In some embodiments, a biospecimen tested within a diagnostic system maybe blood. In some embodiments, well analyses of blood componentscontaining pathogenic biological molecules can improve functions ofdiagnostics (e.g., emerging infectious diseases), and, thus, adiagnostic system may be configured to simultaneously examine pathogenicDNA, RNA, and/or protein in blood. In other embodiments, well analysesof blood components containing biological molecules can provide usefulinformation with respect to pre-natal, oncological or otherapplications, and, thus, a diagnostic system may be aimed tosimultaneously examine DNA/RNA/protein in blood. Example capabilitiesand components of a diagnostic system may include a sample collectionmodule, sample separation module (e.g., plasma separation), a lysismodule, a sample preservation module, detection/analysis module (e.g.,modules for detecting/analyzing proteins or nucleic acids), and/or anintegrated read-out module.

A point of care system of the present disclosure can include a chip thatcomprises a plurality of sensors, as described elsewhere herein (e.g.,impedance measurement sensors). The chip can be part of a housing orcartridge that may be integrated with other modules for sample retrievaland processing, as well as a computer processor or other logic forfacilitating sample processing and analysis.

As an alternative, the chip can be part of a housing or cartridge thatis separate from other components of the system. For example, the chipcan be part of a cartridge that can be inserted into or removed from ahousing containing the computer processor (or other logic), such asthrough a port. The housing in such a case can include other componentsfor sample processing, such as a sample retrieval port and a fluid flowsystem (e.g., pump) or apparatus, which can be brought in fluidcommunication with the chip when the cartridge has been inserted intothe housing. In some embodiments, a fluid flow apparatus or system maybe or may comprise a microfluidic device.

FIG. 78 shows an example integrated sample analysis system. The systemcomprises a sample collection module 7801, sample processing module7802, a sensing module 7803 comprising an array of sensors, fluid flowmodule 7804 comprising a fluid flow system, and other modules 7805 and7806 all integrated in a housing 7807. The housing 7807 can be acartridge. The modules can be in fluid communication with one anotherthrough channels in the housing 7807, or in one or more layers adjacentto the housing 7807. The system of FIG. 78 can be a point of caresystem.

Preservation of Biological Samples

A diagnostic system may be a microfluidic, powerless, and/orreagentless. In some embodiments, a diagnostic system may include asample preservation module that may not require medical training, andmay stably store DNA, RNA, proteins, etc. for extended periods, such asup to about five days, fifteen days, one month, two months, threemonths, four months, five months, or more at room temperature. In someembodiments, a sample preservation module can comprise sugar and silicagel (e.g., Trehalose, sucrose, sol-gel, etc.) matrixes for thepreservation of biological targets of interest. Moreover, a samplepreservation module may make use of bio-inspired micropore evaporationmicrofluidics that can enhance drying processes useful for samplepreservation.

In some embodiments, a sample preservation module may separately storeblood components, such as, for example, serum and whole blood. Eachspecies can be purified via monolithic filters so that DNA/RNA/Proteinscan be stored in separate storage chambers. For serum, blood cells maybe filtered (e.g., filtered via a sample preparation module thatincludes filtration capabilities), and pathogens in serum may be lysed.In some embodiments, serum may be preserved for the lower interferenceof false signals for downstream analysis. The process may be the samefor whole blood, with the exclusion of filtration. Preservation of wholeblood can be of interest because some pathogens such as, for example,Plasmodium parasites (malaria) and HIV virus, can replicate in bloodcells and can have high counts.

In some embodiments, monolithic filters may be used to selectively allowonly lysate DNA, RNA, or proteins to flow into each storage chamber.These filters may function by negative filtration based on size, charge,and selective degradation enzymes (e.g., RNAase, DNAase, Proteases,etc.). In some embodiments, filtration can provide a convenientpretreated specimen for downstream analysis.

In some embodiments, biospecimens may be preserved using Trehalose sugarbased glassification. The protective effect of Trehalose can stabilizemembranes and lipid assemblies at low levels of hydration that wouldnormally promote their denaturation. The properties of Trehalose canmake it a suitable candidate for sample preservation, including delicatebiological structures in dehydrated forms at ambient temperature. Insome embodiments, other components, such as sucrose and dextran, havebeen shown to be complementary to Trehalose preservation ofbiomolecules.

In some embodiments, silica gels, which are nanoporous sol-gels, mayenhance protein preservation along with Trehalose by removing excesswater content. Silica gel is inorganic and inert, thus it does notchemically affect the quality of the stored samples. Trehalose treatedsamples may be dehydrated to preserve the biomolecules. Silica gel maybe used as a desiccant due to its large surface area and strong affinitywith water.

In some embodiments, sugar-based 3D micropillar structures may becreated in the storage area using material jet printers (e.g., FUJIFILMDimatrix Materials jet). These sugar microstructures can have nanoporesso as to significantly increase reaction surface area forTrehalose-biospecimen stabilization.

In some embodiments, a microporous top membrane structure for the rapiddehydration of biosamples stored in Trehalose may be utilized. A shortduration (e.g., a few minutes) of infrared irradiation may aid in theevaporation of excess water content. Moreover, air drying may be used toaid in dehydrating samples after Trehalose treatment. In someembodiments, the leaf-like micropore structures of a top membrane mayhelp facilitate on-chip drying. Silica gel may be incorporated nearmicropore to assist in desiccation.

In some embodiments, the packing of the entire device may be in a vacuumcapsule that has a silica gel component and may have the ability to beresealed in an airtight fashion. The vacuum capsule may be used inkeeping the a system viable. It may also help to protect on-chiplyophilized reagents from oxidizing. An air-tight seal and silica gelcan be used to isolate chips and keep humidity low to stabilize storageconditions.

In some embodiments, the biomolecule of interest (e.g., DNA, RNA,protein, etc.) may be stored in Trehalose based sugars. The preservedsamples may be compatible with standard downstream analysis techniques,including western blotting, ELISA, PCR, MALDI, conventional massspectroscopy, etc., or other suitable technique.

Fabrication may be via standard industrial techniques for massproduction, such as for example, injection molding and fabrication. Insome embodiments, patterned sugars may be fabricated by 3D printing.

Sample Collection

Systems of the present disclosure may comprise systems or devicescapable of obtaining a sample from a subject, such as for example, ablood sample. In some embodiments, an array of microneedles may beintegrated into the device for a painless medium for conducting bloodfrom the subject to the microfluidic channels of a device.Microfabrication methods may allow for the creation of arrays ofmicroneedles to painlessly withdraw small blood samples. See, e.g., H.J. G. E. Gardeniers et al., J. Microelectromech S 12, 855, and R. K.Sivamani, D. Liepmann, H. I. Maibach, Expert Opin Drug Deliv 4, 19, eachof which is entirely incorporated herein by reference. In someembodiments, the microneedles penetrate up to about 100, 200, 300, 400,500, 600, 700, 1000, or more microns into the dermis of the skin, wherethe microneedles can reach capillaries, but not nerves. In otherembodiments, the depth of penetration may be smaller or greater thatabout 400 microns. Moreover, at lengths such as about 400 microns, asmall diameter array may also permit stretching and compression ofsurrounding tissues, allowing for painless withdrawal of blood from asubject. See, e.g., S. Kaushik et al., Anesth Analg 92, 502, which isentirely incorporated herein by reference.

In some embodiments, a microneedle array may also incorporate a driedanticoagulant coating. The microneedle array may be pre-treated with ananticoagulant to help sustain blood flow to the microfluidic device.

In some embodiments, a sedimentation based sample fractionation systemmay be used in order to harness gravity based differential sedimentationto separate plasma from whole blood. In some embodiments, autonomouspumping through the fractionation system can be based on slow release ofvacuum pressure through nanoporous polymers (e.g., PDMS). An example ofother sedimentation systems that may be employed for use with methods,devices and systems of the present disclosure are described in I. K.Dimov et al., Lab Chip 11, 845, which is entirely incorporated herein byreference.

In some embodiments, sedimentation based separation of red blood cellsand white blood cells may be used to remove mammalian DNA andamplification inhibitors (e.g., hemoglobin). The plasma separated fromthe trenches may then flow downstream, into, for example, aelectrochemical lysis module for the lysing of bacterial, plasmodium,and/or viral pathogens.

In some embodiments, the separation of red blood cells and white bloodcells from the smaller and lighter bacterial, plasmodium, and/or viralcells via differential sedimentation can leverage the large differencein sedimentation rates between the bloods cells and thebacteria/plasmodium/viruses. This may allow for high efficiencyseparation in a microfluidic environment. A trench based filterstructure may be used, wherein the trenches can be placed at regularintervals along the channel to capture the contaminating host cells. Inone example, the channel height may be about 80 μm with deep trenches ofabout 1 mm in height, and a cross sectional area of 24 mm². In someother embodiments, the channel height may be smaller than 80 μm forintegration and smaller sample volume. In other embodiments, where thelarger sample sizes are desired the channel height may be in 100 s of μmor larger. In some cases, the volume can be optimized based on theapplication and the biological sample size (e.g., blood). In someembodiments, tube-less and power-less fluid propulsion systems may beused in a system. The system may include a block of porous material(such as PDMS) that has been degassed (vacuumed) during packaging. Bloodcan be sucked into the microfluidic device due expansion of the poreswithin the PDMS block that cause the re-absorption of air present in theunprimed microfluidic system that drives blood flow into the chip. Thechip may be prepackaged in vacuum bags. Other porous polymers that arecompatible with the device design and easily manufacturable with hotembossing and/or injection molding may be used.

Electrochemical Lysis

In some embodiments, after plasma separation from blood, the plasma maybe transported to a lysis module, such as, for example, an electrolysismodule, in some cases, a tunable hybrid electrolysis module. A tunablehybrid electrolysis module may possess both electroporation andelectrochemical lysis capabilities, which can be used for lysing bothpathogenic and human cells selectively. Selective lysis can allowflexibility of processed sample output for use in downstream assays, aslysate of select pathogens can be obtained. Moreover, electrolysis basedlysis is reagentless, and, thus, does not generally interfere withdownstream assay(s). Moreover, low power operation may also be possiblesince electrical fields can be concentrated in microscale geometries.Furthermore, device complexity may be reduced, as the number of fluidicinputs can be reduced since lysis is completed with electrodes, ratherthan reagents.

In some embodiments, an electric current may be used to generate lytichydroxide ions on-chip that can function as lysis agents in cellmembrane lysis. Hydroxide ions can function as cell lysis agents bycleaving fatty acid groups within cell membrane phospholipids. Hydroxideions can be generated at low voltages (˜2.5 V) and little amounts ofpower (currents ˜10 μA). Higher voltages can be assumed to generatehigher hydroxide concentrations and thus accelerate lysis; however,increasing the voltage to high levels (e.g., above 3V) where electrodedegradation can occur, may not significantly decrease cell lysis time.In some embodiments, the lysis time is typically about 0.5 minutes, 1minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4.0minutes, 4.5 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9minutes, 10 minutes, or more.

For the pathogens of interest, which include bacteria, plasmodium, andviruses, membrane properties may be similar to mammalian cells and,thus, cells can be lysed on-chip. Additionally, electrolysis may notcontaminate sample because excess OH⁻ ions can be quenched downstreamvia recombination with H⁺ ions generated at, for example, an anode. ThepH for lysis of cells can be above 11.2 and some biological species(e.g., DNA) are not damaged at this pH. For example, plasmid extractionis frequently performed at pH levels of 12.0 and above.

In some embodiments, the tunable electrochemical lysis module may beable to selectively lyse and release the genomic content of theplasmodium/viral cells based on a hybrid system of electrochemical lysisand electroporation lysis. Platelet cells present can also be lysed, butthe absence of genomic DNA may minimize contamination concerns.

Plasmodium parasites have lipid membranes, which may be lysed withelectrochemical techniques described herein by generating H+ and OH−ions (similar to mammalian cell lysing). Viruses, however, have proteincapsids and matrixes which may not be easily lysed by just changes inpH. In some embodiments, short pulses of high voltage spikes can be usedto porate viral membranes (electroporation), in conjunction with high pHgenerated by electrochemical lysis (a low constant voltage) lyse theviral membranes. By operating at different regimes (e.g., introducingdifferent amplitudes of pulses and constant voltages), the tunableelectrolysis module may be able to lyse pathogens selectively. In someembodiments, bacterial cells may be selectively lysed. In otherembodiments, other possible biomolecules can be selectively separatedwith similar techniques.

In some embodiments, cell lysis may be performed on-chip using othermeans such as using detergents, high electric fields, mechanical,electroporation techniques, and/or thermal stresses.

Pre-Concentration of DNA

Systems of the present disclosure may comprise systems or devicescapable of enriching species such as nucleic acids, proteins, or otherspecies. Systems of the present disclosure can be used to enrich atleast a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or1000-base nucleic acid sequence (e.g., DNA or RNA) by up to 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, or5000-fold into a concentrated band using bipolar electrodes (BPEs). Insome embodiments, the enrichment can be monitored using electronic orfluorescent-based approach (e.g., fluorescence microscopy). Onceenrichment is initiated, the location of the concentrated band can bemanipulated by changing the velocity of the PDF. The flow rates thatoccur with the chip can be within the PDF rates that enablepre-concentration with BPEs.

Upon liberation of pathogenic biomarkers using electrochemical lysis,enrichment of liberated nucleic acids (e.g., DNA) using bipolarelectrodes (BPEs) integrated within a device may be performed. Whensufficient potential is applied across a buffer filled microchannelcontaining a BPE, faradaic reactions can be induced at its poles.Hydroxide produced by water reduction at the BPE cathode can neutralizebuffer cations resulting in the formation of a depletion zone andconsequently, an electric field gradient. Enrichment of analyte anions(e.g., nucleic acids) may occur at the position on the electric fieldgradient where the velocity due to bulk convection, (nearly uniformalong the length of the channel), is exactly balanced by an equal andopposite electrophoretic velocity (which is a function of location alongthe electric field gradient), where convective flow may be due toelectroosmosis and pressure-driven flow (PDF) combined.

The location on the electric field gradient at which enrichment occurscan be dependent on the electrophoretic mobility (μ_(ep)) of theenriching species. Therefore, species with different μ_(ep) values mayenrich into separate concentrated bands within the same channel.Different DNA oligomers may be separated using this same approach andvalving with cross channels may enable selective capture.

Nucleic Acid and Protein Signal Amplification

Systems of the present disclosure may comprise systems or devicescapable of nucleic acid amplification. Traditionally, nucleic acidamplification-based diagnostics have been performed using PCR. With theemerging need for integrated and portable molecular diagnosticsolutions, PCR reactions have been adapted for microfluidic chips.However, PCR necessitates thermocycling, which adds significant powerconsumption, complexity, and cost to the process. To this end,isothermal nucleic acid amplification schemes may be used, in particularLoop Mediated Isothermal Amplification (LAMP).

LAMP is an amplification process in which DNA of interest is amplifiedusing a set of primers at a constant temperature of 65° C.˜70° C. See,e.g., T. Notomi et al., Nucleic Acids Res 28, E63, which is entirelyincorporated herein by reference. LAMP-based assays may be integratedon-chip using the self-powered degassing method for automated moleculardetection. The assay may allow for visualization by naked-eye and properillumination facilitates fluorescent excitation for quantitativeanalysis. In some embodiments, the reagents and Bst DNA Polymerase maybe lyophilized on-chip for long-term storage. Upon sample loading, theserum reconstitutes the lyophilized reagents and the amplificationprocess begins. In one embodiment, primers for a region of the 16S rDNAgene that is universally conserved among both gram negative and positivebacteria may be used. However, the 16S rDNA gene itself ishypervariable, thus allowing the generation of amplicons that arespecies specific and can be detected downstream using, for example,aptamer and aptazyme probes (discussed below). In some embodiments, asystem may be used for the detection of drug-resistant bacteria. Inother embodiments, a system can detect the conserved and drug-resistantgenomic regions for a number of infectious disease pathogens. In someembodiments, primer sets can be designed for drug-resistant genes suchas those that encode β-lactamase, mecA (methicillin-resistantStaphylococcus aures), and rpoB (Rifampicin-resistant Tuberculosis).

Systems of the present disclosure may comprise systems or devicescapable of detecting proteins. In some embodiments, the system may be adetection platform, integrating biomolecular sensor and actuatorcomponents into a high-throughput microfluidic system. Sensitive proteindetection may be achieved, for example, by way of a specificprotein-aptamer conjugate. Target-specific aptamers can be easilygenerated regardless of immunogenicity or target toxicity. Once aspecific aptamer has been selected and sequenced, unlimited amounts ofthe same aptamer can be synthesized with little effort and investment.See, e.g., A. D. Ellington, J. W. Szostak, Nature 346, 818, which isentirely incorporated herein by reference. Aptamers can be linked withcatalytic oligonucleotide regions to create aptazymes. See, e.g., S.Cho, J. E. Kim, B. R. Lee, J. H. Kim, B. G. Kim, Nucleic Acids Res 33,E177, which is entirely incorporated herein by reference. Upon specificbinding with a target molecule, such as, for example, a cytokine orantibody, the aptamer region may undergo a conformation change that mayactivate the linked catalytic region, leading to a signaling event, asshown in FIG. 79. For example, by conjugating Bst DNA Polymerase to theportion of the aptazyme that undergoes nucleolytic cleavage, thissignaling event may be detected downstream. In some cases, the signalingevent may be amplification of a nucleic acid. In some embodiments,signal amplification is achieved via a LAMP amplification reaction asdescribed elsewhere herein. For example, the combination of aptazymesand LAMP may be referred to as AptaLAMP. A visual signal can enablenaked-eye readout or a quantified signal may be measured via an opticalreader or CMOS-based electronic detection.

An example of an aptazyme approach is shown in FIG. 79. As shown in FIG.79, a bead 7901 may be linked to a nucleic acid 7902 hybridized with aprimer. The bead 7901 may be proximate an aptamer 7904 linked to apolymerase 7903 (e.g., Bst DNA polymerase). A substrate 7095 (e.g., atarget analyte such as a protein, nucleic acid, small molecule, etc.)can bind 7910, with apatmer 7904. Upon binding of substrate 7904, theaptamer functions as an aptazyme an releases 7911 its bound polymerase7903. The released polymerase can bind 7912 to nucleic acid 7902 andextension of the primer of nucleic acid 7902 can commence. Nucleotideincorporation 7906 can be detected using methods described herein (e.g.,detecting a local impedance change), effectively functioning as asignaling event for binding of substrate 7905 to aptamer 7904.

RNA Biomarker Transduction Using RNA Restriction Enzymes

Systems of the present disclosure may comprise systems or devicescapable of detecting RNA. The system may allow for RNA detection withinblood pathogen samples that employs a recently identified class ofendoribonucleases involved in the prokaryotic immune system (see e.g.,S. J. Brouns et al., Science 321, 960, which is incorporated herein byreference in its entirety). In host bacteria, RNA transcripts can bederived from Clustered Regularly Interspaced Short Palindromic Repeats(CRISPRs) that can be processed by these enzymes into shorterCRISPR-derived RNAs (crRNAs). Such crRNAs may be subsequently used totarget and destroy viral nucleic acids in a process. See, e.g., J. vander Oost, M. M. Jore, E. R. Westra, M. Lundgren, S. J. Brouns, TrendsBiochem Sci 34, 401, and M. P. Terns, R. M. Terns, Curr Opin Microbiol,each of which is entirely incorporated herein by reference. Althoughenzymes within this superfamily share common structural and catalyticproperties, their ability to recognize diverse RNA sequences has evolvedin response to rapid bacteriophage evolution. See, e.g., K. S. Makarova,N. V. Grishin, S. A. Shabalina, Y. I. Wolf, E. V. Koonin, Biol Direct 1,7 and V. Kunin, R. Sorek, P. Hugenholtz, Genome Biol 8, which isentirely incorporated herein by reference. As a result, enzymes existthat recognize a large number of distinct RNA sequences—analogous to thediversity of substrate specificity observed among DNA restrictionenzymes. These enzymes may be used, which will be referred to as RNArestriction enzymes (RREs), to develop a simple, low-cost method ofdetecting pathogen RNAs.

For example, in order to obtain a large orthogonal set of proteins forspecific and selective RNA sequence detection, CRISPR transcripts havebeen processed in Escherichia coli, Pyrococcus furiosus, and Pseudomonasaeruginosa. In each case, a single enzyme responsible for this activityhas been identified: CasE in E. coli (see, e.g., S. J. Brouns et al.,Science 321, 960), Cas6 in P. furiosus (see, e.g., J. Carte, R. Y. Wang,H. Li, R. M. Terns, M. P. Terns, Gene Dev 22, 3489), and Csy4 in P.aeruginosa (see, e.g., R. E. Haurwitz, M. Jinek, B. Wiedenheft, K. H.Zhou, J. A. Doudna, Science 329, 1355). These enzymes are specific fortheir own associated crRNA sequence and do not cleave heterologousCRISPR RNAs (see, e.g., R. E. Haurwitz, M. Jinek, B. Wiedenheft, K. H.Zhou, J. A. Doudna, Science 329, 1355). However, crystal structures ofCas67 (see, e.g., R. Wang, G. Preamplume, M. P. Terns, R. M. Terns, H.Li, Structure 19, 257), CasE (see, e.g., Y. Kurosaki et al., J VirolMethods 141, 78), and Csy4 (see, e.g., R. E. Haurwitz, M. Jinek, B.Wiedenheft, K. H. Zhou, J. A. Doudna, Science 329, 1355) revealed thatthey comprise similar protein folds, indicating an evolutionarilyconserved architecture. Furthermore, co-crystal structures of these RREsbound to their crRNA targets highlighted mechanisms of substraterecognition that impart the high degree of sequence specificity criticalto their application. Because CRISPR systems in different organismscontain distinct RNA sequences that constitute the sites of RREprocessing, the diversity of RNA recognition may be large. RNArecognition sites may be minimal (e.g., 5-10 base pairs) and can bereconstituted by two oligonucleotides hybridized in trans. Theseattributes may lend themselves well to the analysis of diverse RNAmolecules.

In some embodiments, pathogen RNAs may be recognized and detected withina system module. In the presence of exogenously supplied,nuclease-resistant oligonucleotides, pathogen RNAs in human bloodsamples—if present—can efficiently base-pair with their complementarysequence found in guide oligonucleotides associated with the module.This hybridization can generate double-stranded RNAs that are fullycompetent substrates for endoribonucleolytic cleavage by RREs. Takingadvantage of the limited interactions that RREs exhibit with nucleicacid downstream of the cleavage site, Bst DNA Polymerase reporter can bechemically conjugated to the 3′ end of guide oligonucleotides. See,e.g., R. E. Haurwitz, M. Jinek, B. Wiedenheft, K. H. Zhou, J. A. Doudna,Science 329, 1355, which is entirely incorporated herein by reference.These reporters may be released after RRE-mediated cleavage, resultingin a spectrophotometric signal that is easily detected.

RREs can be chemically tethered to the surface of the microfluidics chipusing standard protein conjugation techniques. A positive signal withinthis approach may only be generated if the target RNA sequence exists inthe pathogen sample; if no such sequence is present, the double-strandedsubstrate is not formed and the RRE remains inert. By multiplexingthrough use of multiple RREs and guide oligonucleotides, each finelytuned to probe for a specific pathogen RNA sequence, this approach canenable sensitive yet accurate RNA biomarker detection.

Surface Functionalization with Nucleic Acids

In some embodiments, in order to integrate LAMP, AptaLAMP, and RREdetection on-chip, various types of biomolecules may be patternedon-chip. Methods described herein may be used to covalently immobilizenucleic acids (e.g., DNA) directly onto a microchannel surface, aconfiguration which may be useful, for example, for an enzyme-linked DNAhybridization assay. In some embodiments, DNA can be directly attachedto PDMS microfluidic channels, and the use of these PDMS-immobilizedcapture probes can be used for further immobilization of proteins. Suchan approach may be used with other approaches for controlling surfaceproperties of PDMS and the use of surface modifications forimmobilization of DNA, RNA, and proteins, such as those described in D.Liu, R. K. Perdue, L. Sun, R. M. Crooks, Langmuir 20, 5905, which isentirely incorporated herein by reference.

In some embodiments, the immobilization of nucleic acid (e.g., DNA) ontoa PDMS surface may involve a plurality of steps which can include:plasma-induced oxidation of the PDMS surface, functionalization of theoxidized surface with a silane coupling agent bearing a distal thiolgroup (mercaptopropylsilane, MPS), and subsequent reaction of the thiolgroups with acrylamide-modified DNA. The silanization step can becarried out using a vapor-phase reaction method. The plasma-treated PDMSmay be exposed to acid (e.g., HCl) vapor before the MPS vapor, as theacid can act as a catalyst that increases the rate of MPS immobilizationon the PDMS surface. Subsequent exposure of the PDMS-linked DNA to itsbiotinylated complement can provide a platform for immobilization of aprotein (e.g., alkaline phosphatase (AP)). PDMS immobilization ofspecies can be compatible a variety of species, including thosedescribed herein. In some cases, PDMS immobilization can provides ameans for immobilizing any suitable oligonucleotide orstreptavidin-modified protein onto a PDMS surface.

Nucleic Acid Patterning and Replication for Mass Fabrication

In some embodiments, a method for parallel replication of DNA and RNAmicroarrays of arbitrary size may be used. Other approaches for parallelreplication of DNA and RNA are described in, for example, J. Kim, R. M.Crooks, Anal. Chem. 79, 7267, 8994, which is entirely incorporatedherein by reference.

For DNA arrays, approach can consist of a number of steps, with examplesof such steps described below. For example, a master DNA array may beprepared by covalent immobilization of amine-functionalized DNAtemplates on an epoxy-modified glass substrate. Second, biotinylatedprimer oligonucleotides, consisting of a single sequence, can behybridized to the distal end of the template DNA, and the primers may beextended using a T4 DNA polymerase enzyme. Third, a streptavidin-coatedpoly(dimethylsiloxane) (PDMS) monolith can be brought into contact withthe master array. This may result in binding of the extended,biotinylated primers to the PDMS surface. Fourth, the PDMS substrate canbe mechanically separated from the glass master array. This may resultin transfer of the extended primers to the PDMS surface, and it mayleave the original master array ready to prepare a second replicatearray.

An approach similar to that described above can be used to patternproteins appended with short DNA labels. In this case, a DNA zip codearray may be prepared, and it directs the protein to the specifiedlocation. See, e.g., H. Lin, J. Kim, L. Sun, R. M. Crooks, J Am Chem Soc128, 3268, which is entirely incorporated herein by reference.

Signal Transduction

Systems of the present disclosure may comprise systems or devicescapable of detecting proteins, nucleic acids (e.g., DNA or RNA), orother species either directly or indirectly. Real-time monitoring ofpolymerase reactions are typically performed with fluorescent molecules,which transduce the product of the reaction into an optical signal. Insome embodiments, signal detection methods may be used to monitorpolymerase reactions in real-time, with non-limiting examples thatinclude optical-based and CMOS-based modalities.

In one embodiment, an optical-based method, such as for example, anoptical readout technique may use reporter molecules to generate afluorescent signal. For quantitative readout of the assay, the chip canthen be inserted into an instrument, which can maintain assaytemperature, illuminate the chip, and detect fluorescence emission fromthe reaction chambers using an array of phototransistors. The instrumentmay be designed to perform its functions without the use of costlyoptical components and without the need for alignment or focusing.

In one embodiment, the instrument may be automated, for example, amicrocontroller board. In some embodiments, the instrument may feature aUSB interface, a Secure Digital (SD) Flash memory card reader forstoring assay parameters and results, and color touch screen userinterface. In a further embodiment, to run an assay, the microfluidicchip can inserted directly on top of a 4 indium tin oxide (ITO) coatedglass slide, which heats the chip to an appropriate temperature (e.g.,60° C.). In an exemplary embodiment, blue InGaN LEDs (peak=472 nm) canilluminate the chip from its sides through a glass waveguides claddedwith black paint to minimize stray light. The waveguides promote maytotal internal reflection (TIR) of excitation light within the chip.

In some embodiments, the reporter molecules used in a LAMP reaction mayemit green fluorescence (peak excitation=480 nm, peak emission=515 nm).This fluorescence can be detected with a phototransistor locateddirectly underneath each reaction chamber. There may be a small air gapbetween the phototransistor housings and the ITO heater, which helpsensure TIR and prevent feedthrough of the excitation light into thephototransistors. A microcontroller can use such multiplexers to rasterthrough the phototransistor array, selecting one at a time forinterrogation and the entire array can be sampled at specified intervals(e.g., typically every 10 seconds).

In one embodiment, an instrument can be powered by a lithium polymerbattery (e.g., a 3.7 V, 2000 mAh battery). In an exemplary embodiment,the system can run a typical assay in approximately 0.5, 1, 1.5, 2.0,2.5, 3.0, 3.5, 4 or more hours and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, or more amp-hours are consumed. An enclosure featuring betterthermal insulation that will greatly reduce this power consumption isenvisioned and this can extend the life of the battery. In someembodiments, the instrument is a fully-integrated, portable instrumentwhich addresses the needs of a small clinical setting.

In some embodiments, impedance biosensors (e.g., complementarymetal-oxide-semiconductor impedance sensors) may be used to probe forbiomolecular binding in real-time. Impedance sensors can be used forimpedance, charge and/or conductivity measurements, such as measurementsacross a particle (e.g., bead), through the particle, across a surfaceof the particle, or locally in or within a fluid environment of theparticle. This technique utilizes the cheap, mass produced semiconductorfabrication processes that have been optimized over the past decades forthe integration of label-free biosensing with point-of-care moleculardiagnostic systems.

In some embodiments, a CMOS nanosensor array may be used for thedetection of both protein and nucleic acids. The geometry of impedancesensors (e.g., two impedance sensors) and their co-localization canallow for dual and independent readouts of the same biomolecular bindingevent. Co-localized sensors may each measure impedance changes as anelectrical ‘signature’ of nucleic acid hybridization or proteininteraction, but via different mechanisms, as shown in FIGS. 80A-80D.One sensor, for example a NanoNeedle, can be an ultra-sensitive,localized impedance biosensor, which can detect local changes inimpedance via electrical current changes. In some embodiments of theNanoNeedle, an active (20 nm) double electrode tip may be in immediatecontact with the reaction solution, resulting in the ability to measureminute changes in resistance down to the aM concentration change level.

In a further embodiment, the second sensor, for example, may beconfigured as a NanoBridge and may function as a double gatedion-sensitive semiconductor sensor based on a depletion mode‘nanoresistor’ and can use electrical current as readout. In someembodiments, the NanoBridge may be fabricated of semiconductor materialwith an optimized engineered doping profile that significantly cansignificantly increase the sensitivity of the system. In contrast to aFET, this device can be always in the “ON” state, and no thresholdvoltage may be needed to turn it to active sensing mode. In addition,signal calibration may not be needed due to the linear I-Vg response atlow Vg. The NanoBridge design may be optimized for maximal ΔI/I. Theresponse can be linear and the linearity of the response can show thatthe design can allow for a measurement of charge induced changes over awide concentration range with low threshold and good signal to noiseratios.

In one embodiment, for the detection of nucleic acid and protein, asilicon oxide surface of a sensor may be directly functionalized withcapture nucleic acid (e.g., DNA) or aptamers using3-aminopropyltriethoxysilane (APTES). APTES can form a monolayer byspecifically and covalently interacting (via formation of a Schiff base)with silicon oxide. The amino group may then be used to immobilize asingle stranded oligonucleotide for DNA hybridization or an aptamer fordirect protein detection. In some embodiments, the method of depositionmay either follow a photolithographic process to generate individuallyaddressable pixels or may be through the application of a mask anddirect ‘implantation’ of the capture molecules. In another embodiment,functionalized sensors can be embedded in microwells that are individualreaction ‘chambers’ to which sample can be delivered throughmicrofluidic channels.

Nucleic Acid Detection Via Hybridization

In one embodiment, nucleic acid (e.g., DNA) hybridization detection fora NanoBridge sensor can have sensitivity down to 3600-4000 molecules, asshown in FIGS. 81A and 81B. In some embodiments, a NanoBridge sensor mayhave sensitivity less than 3600 molecules, for example, down to 2000molecules or less, or 1000 molecules or less, etc. In some embodiments,LAMP based amplification of the conserved 16S rDNA from bacteria may befollowed by individual strain identification via hybridization tosubtype specific regions of the LAMP generated amplicons. The ampliconscan be detected via the specific capture/hybridization probes onfunctionalized sensors. Each specific binding event of a complementaryDNA strand may increase negative surface charge and result in ameasureable increase in conductance.

Methods for Protein Detection

In some embodiments, the use of the CMOS nano sensors (e.g., an exampleshown in FIG. 82) can allow for the direct detection of proteins ofinterest using aptamer functionalized sensors. The NanoNeedle can detectantibody-protein binding events down to the aM concentration. In someembodiments, the detection of proteins may be achieved by two exemplarymethods 1) direct aptamer functionalization of the sensor surface anddetection of the interaction with its specific target protein and 2) useof the aptaLAMP amplification methods by detection of downstreamsecondary amplicons generated upon protein detection.

In one embodiment, the device may be a disposable chip that contains thesensors, microfluidic and electric wiring components to allow thedetection of protein biomarkers (host and parasite) and correspondingDNA species of interest for one to multiple samples. In someembodiments, a device may be modular and can be configured quickly toincorporate capture molecules for new biomolecules of interest.

In an exemplary embodiment, a sensitive, accurate dual CMOS electronicnanosensor array embedded within a micro-channel structure may be usedto detect an impedance change resulting from protein or nucleic acidbinding in real time. These electrical nano-biosensors may generate datain real time, rely on fabrication processes long optimized in thesemiconductor industry, do not require expensive labeling reagents, anddo not require expensive optical readout systems.

USB Compatible Interface

Systems of the present disclosure may comprise systems or devicescapable of detecting proteins. In one embodiment, lysing andelectrokinetic pre-concentration may be designed to be within theoperational range of USB 2.0 specifications (e.g., under 5 V and 500 mVloading). This can enable plug and play USB capabilities with adownstream reader. In some embodiments, utilizing common peripheralssuch as mobile phones/laptops/desktops and PDAs to become potentialreaders and power supply sources of a device is envisioned. Moreover,standard commercial batteries may also be able to power a device.

Fabrication

In some embodiments, devices can be composed of three layers polymericmaterial such as polystyrene. The bottom layer, for example, may be ITOor graphite electrodes coated onto plastic. The middle layer, forexample, may contain any desired fluidic channels. The middle layer mayalso have microchannels and trenches on both sides. Such a configurationcan be achieved by sandwiching two molds on each side during injectionmolding. The top layer may simply be a flat plastic sheet to seal themicrochannels. In one embodiment, a device can be manufactured withplastics and/or with the aid of common mass production techniques suchas injection molding and semiconductor processes (electrodespatterning).

Integration Approach with Other Devices

In some embodiments, a device or system may utilize an embeddedmicroprocessor (e.g., PC/104+Linux operating system) for ease ofre-configurability and programming. Moreover, a device may be suitablefor interfacing via USB or any of several wireless protocols forcommunication and display of results on an external laptop or otherdevice.

Wireless System Integration for Networked Readout

The system may enable multiplexed, simultaneous readout from specimencollected with multiple devices. Wireless communication can enableelectronic transmission for diagnostic interpretation, such as, forexample, by a remote physician.

Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 83 shows acomputer system 8301 that is programmed or otherwise configured toreceive, store, and analyze data output from the integrated microfluidicdevice. The computer system 8301 can regulate various aspects of dataanalysis and storage of the present disclosure, such as, for example,using a base-calling algorithm for sequencing analysis or interfacingwith a cloud-based platform for storage of data associated withexperimental runs.

The computer system 8301 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 8305, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 8301 also includes memory or memorylocation 8310 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 8315 (e.g., hard disk), communicationinterface 8320 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 8325, such as cache, othermemory, data storage and/or electronic display adapters. The memory8310, storage unit 8315, interface 8320 and peripheral devices 8325 arein communication with the CPU 8305 through a communication bus (solidlines), such as a motherboard. The storage unit 8315 can be a datastorage unit (or data repository) for storing data. The computer system8301 can be operatively coupled to a computer network (“network”) 8330with the aid of the communication interface 8320. The network 8330 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 8330 insome cases is a telecommunication and/or data network. The network 8330can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 8330, in some cases withthe aid of the computer system 8301, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 8301 tobehave as a client or a server.

The CPU 8305 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 8310. Examples ofoperations performed by the CPU 8305 can include fetch, decode, execute,and writeback.

The storage unit 8315 can store files, such as drivers, libraries andsaved programs. The storage unit 8315 can store programs generated byusers and recorded sessions, as well as output(s) associated with theprograms. The storage unit 8315 can store user data, e.g., userpreferences and user programs. The computer system 8301 in some casescan include one or more additional data storage units that are externalto the computer system 8301, such as located on a remote server that isin communication with the computer system 8301 through an intranet orthe Internet.

The computer system 8301 can communicate with one or more remotecomputer systems through the network 8330. For instance, the computersystem 8301 can communicate with a remote computer system of a user(e.g., subject, researcher, or healthcare provider). Examples of remotecomputer systems include personal computers (e.g., portable PC), slateor tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones,Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®),or personal digital assistants. The user can access the computer system8301 via the network 8330.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 8301, such as, for example, on thememory 8310 or electronic storage unit 8315. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 8305. In some cases, thecode can be retrieved from the storage unit 8315 and stored on thememory 8310 for ready access by the processor 8305. In some situations,the electronic storage unit 8315 can be precluded, andmachine-executable instructions are stored on memory 8310.

The code can be pre-compiled and configured for use with a machine havea processer adapted to execute the code, or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the computersystem 8301, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such memory (e.g., read-only memory, random-access memory,flash memory) or a hard disk. “Storage” type media can include any orall of the tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 8301 can include or be in communication with anelectronic display that comprises a user interface (UI) for providing,for example, raw data as well as graphs and charts associated with anexperimental run. Examples of UI's include, without limitation, agraphical user interface (GUI) and web-based user interface.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for nucleic acid sequencing, comprising:(a) directing a plurality of particles onto an array of sensors, whereinan individual particle among said plurality of particles comprises atemplate nucleic acid molecule coupled thereto, wherein said arraycomprises a plurality of sensors, wherein an individual sensor amongsaid plurality of sensors comprises a transmitter electrode and areceiver electrode, which transmitter electrode or receiver electrode iscoupled to at least another individual sensor among said plurality ofsensors; (b) positioning said individual particle adjacent to saidindividual sensor; (c) performing a primer extension reaction on saidtemplate nucleic acid molecule coupled to said individual particle atsaid individual sensor; and (d) during or subsequent to performing saidprimer extension reaction, measuring a signal that is indicative of achange in impedance between said transmitter electrode and receiverelectrode.
 2. The method of claim 1, wherein said at least anotherindividual sensor is directly adjacent to said individual sensor.
 3. Themethod of claim 1, wherein said at least another individual sensor isseparated from said individual sensor by one or more intermediatesensors of said array of sensors.
 4. The method of claim 1, wherein saidindividual particle is positioned at said individual sensor such thatsaid transmitter electrode and receiver electrode are electricallycoupled to a Debye layer of said individual particle.
 5. The method ofclaim 1, wherein said transmitter electrode and receiver electrode areelectrically isolated in the absence of said individual particlepositioned adjacent thereto.
 6. The method of claim 5, wherein in (b),said individual particle is positioned adjacent to said transmitterelectrode and receiver electrode, thereby bringing said transmitterelectrode in electrical communication with said receiver electrode. 7.The method of claim 1, wherein said transmitter electrode or receiverelectrode, but not both, is shared with said at least another individualsensor.
 8. The method of claim 1, further comprising amplifying saidtemplate nucleic acid molecule prior to (c).
 9. The method of claim 8,wherein said template nucleic acid molecule is amplified while saidindividual particle is held at said individual sensor.
 10. The method ofclaim 1, wherein said individual particle is positioned adjacent to saidindividual sensor using a magnetic field.
 11. The method of claim 1,wherein (d) comprises measuring a signal that is indicative of a changein impedance across (i) said individual particle or (ii) a fluidenvironment comprising said individual particle.
 12. The method of claim1, wherein both of said transmitter electrode and said receiverelectrode are shared with another individual sensor among said pluralityof sensors.